JP2019516735A - Spirolactam NMDA receptor modulators and uses thereof - Google Patents

Abstract

Disclosed are compounds having enhanced potency in modulating the activity of the NMDA receptor. Such compounds may be used in the treatment of pathologies such as depression and related disorders. Also disclosed are other pharmaceutically acceptable delivery forms of compounds, including orally administrable formulations and intravenous formulations.

Description

This application claims the benefit of priority to and the benefit of US Provisional Patent Application No. 62 / 338,767, filed May 19, 2016, the contents of which are incorporated by reference. It is incorporated herein in its entirety.

  The N-methyl-d-aspartate ("NMDA") receptor is a postsynaptic ionotropic receptor that responds, inter alia, to the excitatory amino acids glutamate and glycine as well as the synthetic compound NMDA. The NMDA receptor regulates the entry of both divalent and monovalent ions into postsynaptic neurons via receptor related channels (Foster et al., Nature 1987, 329: 395-396; Mayer et al. , Trends in Pharmacol. Sci. 1990, 11: 254-260). The NMDA receptor is thought to be involved in the designation of neuronal structure and synaptic connections during development and may be involved in experience-dependent synaptic modification. In addition, NMDA receptors are also thought to be involved in long-term potentiation and central nervous system disorders.

  NMDA receptors play a large role in synaptic plasticity underlying many higher cognitive functions such as memory acquisition, memory retention and learning, and in the perception of specific cognitive pathways and pain (Collingridge et al., The NMDA). Receptor, Oxford University Press, 1994). Furthermore, certain properties of the NMDA receptor suggest that it may be involved in the information processing of the brain underlying consciousness itself.

The NMDA receptor is of particular interest because it appears to be involved in a wide variety of CNS disorders. For example, during a cerebral ischemic condition caused by stroke or trauma, excess or excitatory amino acid glutamate is released from damaged or deficient neurons. This excess glutamate binds to the NMDA receptor and opens up its ligand-gated ion channel, then calcium influx raises the concentration of intracellular calcium and activates the biochemical cascade, leading to protein degeneration and cell death. Bring. This phenomenon is known as excitotoxicity and is also thought to be involved in neurological damage associated with hypoglycemia and other disorders ranging from cardiac arrest to epilepsy. In addition, there are preliminary reports showing that it is equally involved in Parkinson's disease, Parkinson's disease and Parkinson's disease related pathologies such as dyskinesia and L-dopa induced dyskinesia, as well as chronic neurodegeneration of Alzheimer's disease. Activation of the NMDA receptor has been shown to be involved in post-stroke spasm, and certain models of epilepsy have been shown to require activation of the NMDA receptor for seizure development. In addition, blocking the NMDA receptor Ca ⁺⁺ channel with the animal anesthetic PCP (phencyclidine) causes a psychotic condition similar to schizophrenia in humans, thus the neuropsychiatric involvement of the NMDA receptor Recognized (reviewed in Johnson, K. and Jones, S., 1990). In addition, NMDA receptors are thought to be involved in certain types of spatial learning.

The NMDA receptor is believed to consist of multiple proteins embedded in the postsynaptic membrane. Of the subunits so far discovered, the first two types form loops to form large extracellular domains, Ca ⁺⁺ permeable pores or channels, which are thought to contain most of the allosteric binding site And forms a plurality of transmembrane regions and carboxyl terminal regions that are folded. The binding of various ligands to the domain (allosteric site) of a protein present on the extracellular surface regulates channel gating. Binding of this ligand is believed to affect conformational changes throughout the structure of the protein, which ultimately causes the channel to open, partially open, partially close or close It is reflected in things.

The NMDA receptor compounds can exert dual (agonist / antagonist) effects on the NMDA receptor through allosteric sites. These compounds are usually referred to as "functional partial agonists". When a major site ligand is present, a partial agonist replaces a portion of that ligand, thus reducing the Ca ⁺⁺ flux through the receptor. When the major site ligand is absent or its concentration is reduced, the partial agonist acts to increase the Ca ⁺⁺ flux through the receptor channel.

  There is still a need in the art for new more specific / strong compounds that can bind to the glycine binding site of the NMDA receptor and provide medicinal benefits. Furthermore, in the medical field there is still a need for such compounds in orally deliverable form.

The present disclosure provides compounds that may be NMDA modulators, such as partial agonists of NMDA. More specifically, the present disclosure relates to the formula:
(I)
Wherein R ^b is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano and C ₁ -C ₆ alkyl, and R ¹ is selected from the group consisting of hydrogen, halogen and C ₁ -C ₆ alkyl, And R ² is selected from the group consisting of hydrogen, halogen and C ₁ -C ₆ alkyl)
Or a stereoisomer, an N-oxide and / or a pharmaceutically acceptable salt thereof.

  Also provided herein are pharmaceutical compositions comprising the disclosed compounds and a pharmaceutically acceptable excipient. Such compositions may be suitable for oral or intravenous administration to a patient.

  In some embodiments, the compounds described herein bind to the NMDA receptor subtype. In some embodiments, the compounds described herein bind to one subtype and do not bind to another subtype.

  In another aspect, autism, anxiety, depression, bipolar disorder, attention deficit disorder, attention deficit hyperactivity disorder (ADHD), schizophrenia, psychotic disorder, psychotic disorder, social withdrawal, obsessive compulsive disorder , Phobia, post-traumatic stress syndrome, behavioral disorder, impulse control disorder, substance abuse disorder, sleep disorder, memory disorder, learning disorder, urinary incontinence, multiple system atrophy, progressive supranuclear palsy, Friedrich ataxia , Down syndrome, fragile X syndrome, tuberous sclerosis, olivary cerebellar atrophy, cerebral palsy, drug-induced optic neuritis, ischemic retinopathy, diabetic retinopathy, glaucoma, dementia, AIDS dementia, Alzheimer's disease, Huntington Chorea, spasms, myoclonus, muscle spasms, Tourette's syndrome, epilepsy, cerebral ischemia, stroke, brain tumor, traumatic brain injury, cardiac arrest, myelopathy, spinal cord injury, peripheral neuropathy, fibromyalgia Treating a condition selected from the group consisting of acute neuropathic pain and chronic neuropathic pain, there is provided a method in a patient in need thereof. Such methods may comprise administering to the patient a pharmaceutically effective amount of the disclosed compound or a pharmaceutically acceptable salt, stereoisomer, N-oxide and / or hydrate thereof.

  In some embodiments, the method comprises treating depression. For example, depression may be caused by major depressive disorder, dysthymic disorder, psychotic depression, postpartum depression, seasonal affective disorder, bipolar disorder, mood disorder or depression due to a chronic medical condition It may include one or more. In certain embodiments, the method may treat schizophrenia. Such schizophrenia is delusional schizophrenia, disorganized schizophrenia, stress-type schizophrenia, indistinguishable schizophrenia, residual schizophrenia, post-schizophrenia depression or simple schizophrenia Disease.

The average 50 kHz USV data of Compound A in the Positive Emotion Learning (PEL) model is shown. 16 shows enhanced [ ³ H] MK-801 binding to wild type NMDAR2 subtype of Compound A. Figure 5 shows mean plasma concentration-time profiles of Compound A after single intravenous (2 mg / kg) and oral (10 mg / kg) doses in male Sprague Dawley rats. Figure 5 shows mean plasma, brain and CSF concentration-time profiles of Compound A after a single oral (10 mg / kg) dose in male Sprague Dawley rats. The average 50 kHz USV data of Compound B in the PEL model is shown. 16 shows enhanced [ ³ H] MK-801 binding to wild type NMDAR2 subtype of Compound B. Figure 5 shows mean plasma concentration-time profiles of Compound B after single intravenous (2 mg / kg) and oral (10 mg / kg) doses in male Sprague Dawley rats. Figure 5 shows mean plasma, brain and CSF concentration-time profiles of Compound B after a single oral (10 mg / kg) dose in male Sprague Dawley rats. The results of in vitro MN, Ames and hERG assays of Compound A are shown. The results of in vitro MN, Ames and hERG assays of Compound B are shown. 7 shows reverse blast-induced cognitive impairment measured by PEL for Compound A. FIG. 23 shows reverse blast-induced cognitive impairment measured by PEL for Compound B. 16 shows the results of a novelty-induced anorexia (NIH) test in rats for Compound A. 16 shows the results of a novelty-induced anorexia (NIH) test in rats for Compound B. 17 shows the analgesic effect of Compound A. The analgesic effect of Compound B is shown. The result of the Porsolt forced swimming test of compound A is shown. The result of the Porsolt forced swimming test of compound B is shown.

  The present disclosure relates generally to compounds capable of modulating NMDA, such as NMDA antagonists or partial agonists, and compositions and / or methods using the compounds of the present disclosure. It should be understood that compounds of the present disclosure may modulate other protein targets and / or NMDA receptor subtypes.

The term "alkyl" as used herein refers to a saturated linear or branched hydrocarbon, such as, for example, C ₁ -C ₆ alkyl, C ₁ -C ₄ alkyl and C ₁ -C ₃ , respectively herein. It refers to a linear or branched group having 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms, which is called alkyl. For example, "C ₁ -C ₆ alkyl" refers to straight or branched chain saturated hydrocarbon containing from 1 to 6 carbon atoms. Examples of C ₁ -C ₆ alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl and neopentyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1- Butyl, 3-methyl-2-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl Pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-Butyl, pentyl, isopentyl, neopentyl and hexyl can be mentioned.

  The term "cyano" as used herein refers to the radical -CN.

  The terms "halo" or "halogen" as used herein refer to fluoro (F), chloro (Cl), bromo (Br) and / or iodo (I).

  The terms "hydroxy" and "hydroxyl" as used herein refer to the radical -OH.

  The term "compound" as used herein is otherwise understood in connection with the present specification, or one particular form of the compound (ie the compound itself, its particular stereoisomer and / or its The compound itself and pharmaceutically acceptable salts, hydrates, esters thereof unless specifically limited to isotopically labeled compounds, or pharmaceutically acceptable salts, hydrates, esters or N-oxides) And N-oxides, including the various stereoisomers and their isotopically labeled forms. It is to be understood that the compounds may refer to pharmaceutically acceptable salts, or hydrates, esters or N-oxides of stereoisomers and / or isotopically labeled compounds of the compound.

  The term "moiety" as used herein refers to a portion of a compound or molecule.

  The compounds of the present disclosure may contain one or more chiral centers and / or double bonds and may therefore exist as geometric isomers and stereoisomers such as enantiomers or diastereomers. As used herein, the term "stereoisomer" consists of all geometric isomers, enantiomers and / or diastereomers of a compound. For example, where a compound having a particular chiral center is indicated, a compound that is indicated without such chirality at that chiral center and other chiral centers of the compound is within the scope of the present disclosure (ie, For example, a compound shown in two dimensions with a "flat" or "linear" bond rather than a three-dimensional with a solid or dashed wedge bond. Stereospecific compounds may be designated as the symbols "R" or "S" depending on the configuration of substituents around a stereogenic carbon atom. The present disclosure includes all the various stereoisomers of these compounds and their mixtures. A mixture of enantiomers or diastereomers may be designated as “(±)” in the nomenclature, but one skilled in the art will understand that the structure may implicitly exhibit a chiral center. Unless otherwise indicated, it is understood that the chemical structure, eg, a diagrammatic representation of the general chemical structure, encompasses all stereoisomers of the specified compound.

  As discussed herein, compounds of the present disclosure may have multiple chiral centers. Each chiral center may independently be R, S or any mixture of R and S. For example, in some embodiments, the chiral centers have a R: S ratio of about 100: 0 to about 50:50 ("racemate"), about 100: 0 to about 75:25, about 100: 0 About 85:15, about 100: 0 to about 90:10, about 100: 0 to about 95: 5, about 100: 0 to about 98: 2, about 100: 0 to about 99: 1, about 0: 100 to 50: 50, about 0: 100 to about 25: 75, about 0: 100 to about 15: 85, about 0: 100 to about 10: 90, about 0: 100 to about 5: 95, about 0: 100 to about 5 2: 98, about 0: 100 to about 1: 99, about 75: 25 to 25: 75 or about 50: 50. Formulations of compounds of the present disclosure that contain one or more isomers (ie, R and / or S) in a larger ratio have enhanced therapeutic characteristics as compared to racemic formulations of the disclosed compound or mixture of compounds. It can.

  The individual enantiomers and diastereomers of the compounds of the present disclosure may be prepared by synthesis from commercially available starting materials that contain asymmetric centers or stereocenters, or after preparation of the racemic mixture, by resolution methods well known to the person skilled in the art be able to. Examples of such resolution methods include (1) combining a mixture of enantiomers with a chiral auxiliary, separating the resulting mixture of diastereomers by recrystallization or chromatography, and optically pure from the auxiliary (2) formation of a salt using an optically active resolving agent, (3) a method of directly separating a mixture of optical enantiomers on a chiral liquid chromatography column, or (4) a stereoselective chemical reagent Or kinetic resolution using enzyme reagents. The racemic mixture can also be resolved into its component enantiomers by well known methods such as chiral phase gas chromatography or crystallization of the compound in chiral solution. Stereoselective synthesis, which is a chemical or enzymatic reaction in which a single reactant forms a mixture of unequal stereoisomers in the process of formation of new stereocenters or conversion of existing stereocenters, is well known in the art It is. Stereoselective synthesis encompasses both enantioselective and diastereoselective transformations. See, for example, Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim, 2009.

Geometrical isomers resulting from the arrangement of substituents around a carbon-carbon double bond or the arrangement of substituents around a cycloalkyl or heterocycle may also be present in the compounds of the present disclosure. symbol

Represents a bond which may be a single bond, a double bond or a triple bond as described herein. The substituents around the carbon-carbon double bond are indicated to be in the "Z" or "E" configuration, where the terms "Z" and "E" are used according to the IUPAC standard. Unless otherwise stated, structures in which double bonds are recited encompass both "E" and "Z" isomers.

  Alternatively, a substituent around a carbon-carbon double bond can be referred to as "cis" or "trans", where "cis" refers to a substituent on the same side of the double bond, and "trans" is And a substituent on the opposite side of the double bond. The arrangement of substituents around the carbocycle may also be designated as "cis" or "trans". The term "cis" refers to a substituent on the same side of the plane of the ring, and the term "trans" refers to a substituent on the opposite side of the plane of the ring. A mixture of compounds is designated "cis / trans" when the substituents are located on both the same and the opposite side of the face of the ring.

The present disclosure is also identical to the compounds described herein except that one or more atoms are replaced with an atom having an atomic mass or mass number that differs from the atomic mass or mass number normally found in nature. Included are isotopically labeled compounds. Examples of isotopes which can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ² H (“D”), ³ H, ¹³ C, ^{14 respectively.} C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl and the like. For example, in the compounds described herein, one or more H atoms are replaced by deuterium.

Certain isotopically labeled compounds (eg, those labeled with ³ H and ¹⁴ C) may be useful in compound and / or substrate tissue distribution assays. Tritiated (i.e., ³ H) and carbon-14 (i.e., ¹⁴ C) isotopes are particularly preferred for their ease of preparation and high detection sensitivity. In addition, substitution with heavier isotopes such as deuterium (i.e., ² H) provides certain therapeutic benefits (e.g., increased in vivo half life or decreased required dose) by increased metabolic stability. In some circumstances such substitution may be preferred. Isotopically labeled compounds can generally be prepared by replacing non-isotopically labeled reagents with isotopically labeled reagents according to methods similar to those disclosed herein, eg, in the experimental section.

  As used herein, the terms "pharmaceutically acceptable" or "pharmaceutically acceptable" refer to adverse reactions, allergic reactions or other inconveniences when administered to animals or humans as appropriate. Refers to compounds, molecular entities, compositions, substances and / or dosage forms that do not cause such reactions. When administered to humans, the formulation must meet the standards for sterility, pyrogenicity, general safety and purity required by FDA biologics agency standards.

  The expression "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, coatings, isotonic agents, and the like compatible with pharmaceutical administration. Refers to absorption delaying agents and the like. Pharmaceutically acceptable carriers may include phosphate buffered saline, water, emulsions (eg, oil / water or water / oil emulsions) and various types of wetting agents. The composition may also include stabilizers and preservatives.

  The phrase "pharmaceutical composition" as used herein refers to a composition comprising at least one compound disclosed herein formulated with one or more pharmaceutically acceptable carriers. . The pharmaceutical composition may also contain other active compounds that provide adjunct, additional or enhanced therapeutic function.

  As used herein, the terms "individual", "patient" and "subject" are used interchangeably and are preferably mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs , Any animal including cattle, sheep, horses or primates, more preferably humans. The compounds described in the present disclosure can be administered to mammals such as humans, but animals that require veterinary treatment, such as domestic animals (eg, dogs, cats etc.), agricultural animals (eg, cattle, etc.) It can also be administered to other mammals such as sheep, pigs, horses, etc.) and experimental animals (eg, rats, mice, guinea pigs etc.). The mammal to be treated by the method described in the present disclosure is preferably a mammal, for example, for which treatment of pain or depression is desired.

  The term "treatment" as used herein includes any effect that results in the amelioration of the condition, disease, disorder or the like, including one or more of its symptoms, eg, alleviation, reduction, modulation, amelioration or loss. . The treatment may be to cure, ameliorate or at least partially ameliorate the disorder.

The term "disorder", unless indicated otherwise, refers to the terms "disease", "pathology" or "disease" and is used interchangeably with them.
The term "modulation" as used herein refers to and includes antagonism (eg, inhibition), agonism, partial antagonism and / or partial agonism.

  As used herein, the expressions "pharmaceutically effective amount" and "therapeutically effective amount" refer to a tissue, system, animal or human biological or medical care sought by a researcher, veterinarian, physician or other clinician. Refers to an amount of a compound (eg, a disclosed compound) that causes a response. The compounds described in the present disclosure can be administered in therapeutically effective amounts to treat disease. The therapeutically effective amount of the compound may be that amount necessary to obtain the desired therapeutic and / or prophylactic effect, such as an amount that results in the amelioration of the symptoms of a disease or disorder such as depression.

  The expression "pharmaceutically acceptable salt" as used herein refers to salts of acidic or basic groups which may be present in the compounds of the disclosure and / or which may be used in the compounds of the disclosure. Point to. Pharmaceutically acceptable salts (eg, acids or bases) of the compounds of the present disclosure can provide the compounds of the present invention, or active metabolites or residues thereof, upon administration to a patient.

  The naturally basic compounds contained in the composition of the present invention can form various salts with various inorganic and organic acids. Non-toxic acid addition salts that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds include non-toxic acid addition salts, ie, but not limited to malate, oxalate, chloride, bromide , Iodide, nitrate, sulfate, hydrogen sulfate, phosphate, perphosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate Pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, gluconate, formate, benzoate, glutamate Salts, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoates (ie 1,1'-methylene-bis- (2-hydro) Shi-3-naphthoate)) including, there is acids which form salts containing pharmacologically acceptable anions. The naturally acidic compounds contained in the compositions of the present invention can form basic salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal salts or alkaline earth metal salts, in particular calcium salts, magnesium salts, sodium salts, lithium salts, zinc salts, potassium salts and iron salts. Compounds containing a basic or acidic moiety contained in the compositions of the invention may also form pharmaceutically acceptable salts with various amino acids. The compounds of the present disclosure may contain both acidic and basic groups, such as one amino group and one carboxylic acid group. In such cases, the compounds may exist as acid addition salts, zwitterions or base salts.

  The compounds disclosed herein may exist as solvated and unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc., and the disclosure relates to solvated and unsolvated forms It is intended to encompass both. In some embodiments, the compound is amorphous. In certain embodiments, the compound is a single polymorph. In various embodiments, the compound is a mixture of polymorphs. In certain embodiments, the compound is in crystalline form.

The term "prodrug" as used herein refers to a compound which is converted in vivo to yield the disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. This conversion may occur by various mechanisms (esterases, amidases, phosphatases, oxidative and / or reductive metabolism, etc.) in various places (such as passage in the intestinal lumen or intestine, in blood or in the liver). Prodrugs are well known in the art (see, eg, Rautio, Kumpulainen et al., Nature Reviews Drug Discovery 2008, 7, 255). For example, if the compound described herein or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, then the prodrug may be a hydrogen atom of a carboxylic acid group ( C ₁ -C ₈ ) alkyl, (C ₂ -C ₁₂ ) alkanoyloxymethyl, 1- (alkanoyloxy) ethyl having 4 to 9 carbon atoms, 1-methyl-1- (having 5 to 10 carbon atoms Alkanoyloxy) -ethyl, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1- (alkoxycarbonyloxy) ethyl having 4 to 7 carbon atoms, 1-methyl-1- having 5 to 8 carbon atoms (Alkoxycarbonyloxy) ethyl, N- (alkoxycarbonyl) aminomethyl having 3 to 9 carbon atoms, 1- having 1 to 10 carbon atoms N (alkoxycarbonyl) amino) ethyl, 3-phthalidyl, 4-crotonolactonyl, .gamma.-butyrolactone-4-yl, di _{-N, N- (C 1 ~C 2} ) alkylamino _(C 2 _-C 3) Alkyl (such as β-dimethylaminoethyl), carbamoyl- (C ₁ -C ₂ ) alkyl, N, N-di (C ₁ -C ₂ ) alkylcarbamoyl- (C ₁ -C ₂ ) alkyl, piperidino- (C ₂ -C ₃₎ alkyl, pyrrolidino - _(C 2 ~C ₃₎ can be alkyl or morpholino _(C 2 ~C ₃₎ esters formed by being replaced with groups such as alkyl.

Similarly, if the compound described herein contains an alcohol function, then the prodrug may be a (C ₁ -C ₆ ) alkanoyloxymethyl, 1-((C ₁ -C ₆ )) of the hydrogen atom of the alcohol group Alkanoyloxy) ethyl, 1-methyl-1-((C ₁ -C ₆ ) alkanoyloxy) ethyl (C ₁ -C ₆ ) alkoxycarbonyloxymethyl, N- (C ₁ -C ₆ ) alkoxycarbonylaminomethyl, succinoyl , (C ₁ -C ₆ ) alkanoyl, α-amino (C ₁ -C ₄ ) alkanoyl, arylacyl, and α-aminoacyl or α-aminoacyl-α-aminoacyl (where each of the α-aminoacyl groups is independently to, naturally occurring L- amino _{acids, P (O) (OH)} 2, P (O) (O (C 1 ~C 6) _{alkyl) 2} or glyceraldehyde May be formed by the replacement by groups such sill being selected from (a radical resulting from the removal of a hydroxyl group of the hemiacetal carbohydrate)).

  When an amine function is incorporated into the compounds described herein, the prodrugs may, for example, be amides or carbamates, N-acyloxyalkyl derivatives, (oxodioxolenyl) methyl derivatives, N-Mannich bases, imines or the like It may be formed by the formation of an enamine. In addition, the secondary amine can be metabolically cleaved to yield a bioactive primary amine, or the tertiary amine can be metabolically cleaved to yield a bioactive primary or secondary amine. For example, Simplicio et al. , Molecules 2008, 13, 519 and references therein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
Throughout the specification, where the compositions and kits are described as having, including, or including specific components, or where the processes and methods are described as having, including, or including specific steps. , The presence of the disclosed compositions and kits consisting essentially of or consisting of the recited components, and the processes and methods according to the present disclosure consisting essentially of or consisting of the recited process steps It is further contemplated that is present.

  In the present application, if an element or component is said to be included in and / or selected from the list of listed elements or components, the element or component is any of the listed elements or components It should be understood that one or more of the elements or components thereof may be selected from the group consisting of two or more of the listed elements or components.

  Furthermore, any combinations of elements and / or features of the compositions or methods described herein may be used in various ways, whether explicitly or implicitly, without departing from the spirit and scope of the present disclosure. Understand that you get. For example, when reference is made to a particular compound, that compound may be used in various embodiments of the compositions of the present disclosure and / or the method of the present disclosure, unless the context otherwise indicates. In other words, although embodiments have been described and shown to enable clear and concise applications to be described and drawn in the present application, without departing from the present teachings and the present disclosure, It is intended and understood that the embodiments may be combined or separated in various ways. For example, it will be understood that all features described and illustrated herein may be applicable to all aspects of the disclosure described and illustrated herein.

  The articles "a" and "an" are used herein to refer to one or more (ie, at least one) of the grammatical object of the article, unless the context is inappropriate. used. By way of example, "element" means one element or more than one element.

  The term "and / or" in the present disclosure is used to mean either "and" or "or" unless otherwise indicated.

  It is to be understood that the expression "at least one" individually includes each of the objects listed after the expression and two or more of the various combinations of the objects listed, unless the context and usage comprehend otherwise . The expression "and / or" in relation to three or more listed objects is to be understood as having the same meaning unless the context indicates otherwise.

  "Include", "includes", "include", "have", "has", "have", "contain", "include" The use of the terms "contains" or "contains" includes their grammatical equivalents, and is generally open-ended and non-limiting, unless specifically understood otherwise from the description or context. It is to be understood that, for example, additional elements or steps not described are not excluded.

  Where the use of the term "about" precedes a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. The term "about" as used herein, unless otherwise indicated or inferred, refers to a ± 10% variation from the nominal value.

  Where percentages are provided with respect to the amount of ingredient or substance in the composition, it is to be understood that the percentages are percentages by weight unless otherwise stated or understood from the context.

  Where a molecular weight is provided and it is not, for example, an absolute value of the polymer, that molecular weight is to be understood as being an average molecular weight, unless otherwise stated or understood from the context.

  It is to be understood that the order of steps or the order for performing a particular operation is not important, as long as the disclosure remains viable. Furthermore, two or more steps or actions can be performed simultaneously.

At various places in the present specification, substituents are disclosed in groups or in ranges. It is specifically intended that the present specification include each and every individual subcombination of such group and scope members. For example, the term _{"C 1-6} alkyl" _{specifically, C 1, C 2, C} 3, C 4, C 5, C 6, C 1 ~C 6, C 1 ~C 5, C 1 _{~C 4, C 1 ~C 3,} C 1 ~C 2, C 2 ~C 6, C 2 ~C 5, C 2 ~C 4, C 2 ~C 3, C 3 ~C 6, C 3 ~C _{5, C 3 ~C 4, C} 4 ~C 6, it is intended to disclose a C 4 -C ₅ and _C 5 -C ₆ individually. As another example, the integer in the range of 0 to 40 is specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 Are intended to be disclosed individually, and integers in the range of 0 to 20 are specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 are intended to be disclosed individually. As a further example, the expression "optionally substituted with 1 to 5 substituents" specifically refers to: 0, 1, 2, 3, 4, 5, 0-5, 0-4, 0 3, 0 to 2, 0 to 1, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4 and 4 to 5 It is intended to disclose individually the chemical groups that may contain the following substituents:

  The use of any and all examples or illustrative words herein, such as “for example,” or “including,” is merely intended to better explain the present disclosure and is claimed The scope of the present disclosure is not limited unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

Further, where a variable does not accompany a definition, the variable is defined as found elsewhere in the disclosure, unless the context indicates otherwise. Furthermore, the definitions of each variable and / or substituent, such as C ₁ -C ₆ alkyl, R ² , R ^b , w etc., occur in the same structure or compound, if they occur more than once in any structure or compound. It may be independent of its definition elsewhere.

  The definitions of variables and / or substituents in the formulas and / or compounds herein encompass multiple chemical groups. The present disclosure provides, for example, i) the definition of variables and / or substituents is a single chemical group selected from the chemical groups described herein, and ii) the definition is described herein. A collection of two or more chemical groups selected from: iii) the compound is defined by a combination of variables and / or substituents, wherein the variables and / or substituents are defined by (i) or (ii) Included embodiments.

  Although various aspects of the disclosure are set forth herein under the headings and / or sections for clarity, all aspects, embodiments or embodiments of the disclosure set forth in one particular section. It is understood that the features are not limited to that particular section, but rather may be applied to any aspect, embodiment or feature of the present disclosure.

Compounds The disclosed compounds have the formula:
(I)
Wherein R ^b is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano and C ₁ -C ₆ alkyl, and R ¹ is selected from the group consisting of hydrogen, halogen and C ₁ -C ₆ alkyl, And R ² is selected from the group consisting of hydrogen, halogen and C ₁ -C alkyl)
Or a stereoisomer, N-oxide and / or pharmaceutically acceptable salt thereof.

In some embodiments, R ¹ is hydrogen. In certain embodiments, R ¹ is C ₁ -C ₆ alkyl, such as methyl (CH ₃ ).

In some embodiments, R ² is hydrogen. In certain embodiments, R ² is C ₁ -C ₆ alkyl, such as methyl (CH ₃ ).

In some embodiments, including any of the embodiments described herein, R ^b is hydrogen. In some embodiments, R ^b is C ₁ -C ₆ alkyl, such as methyl (CH ₃ ).

In various embodiments, the disclosed compounds have the formula:
Or having its stereoisomer, N-oxide and / or pharmaceutically acceptable salts.

  In some embodiments, the compounds are selected from the compounds described in the Examples.

In certain embodiments, the disclosed compounds have the formula:
Or having an N-oxide and / or a pharmaceutically acceptable salt thereof.

In certain embodiments, the disclosed compounds have the formula:
Or having an N-oxide and / or a pharmaceutically acceptable salt thereof.

  The disclosed compounds can provide efficient cation channel opening of the NMDA receptor, and can, for example, bind or associate with the glutamate site of the NMDA receptor to help open the cation channel. The compounds of the present disclosure can be used to modulate (turn on or off) the NMDA receptor through action as an agonist.

  The compounds described herein, in some embodiments, bind to specific NMDA receptor subtypes. For example, the disclosed compounds can bind to one NMDA subtype and can not bind to another NMDA subtype. In certain embodiments, the disclosed compounds may bind to one or more NMDA subtypes, and / or are substantially less (or substantially less than, for example, certain other NMDA subtypes) Not) can have binding activity. For example, in some embodiments, the disclosed compounds (eg, compound A) bind to NR2A and do not substantially bind to NR2D. In some embodiments, the disclosed compounds (eg, compound B) bind to NR2B and NR2D and have substantially lower binding to NR2A and NR2C.

  The compounds described herein may be glycine site NMDA receptor partial agonists. Partial agonists used in this context will be understood to mean that at low concentrations the analogue acts as an agonist and at high concentrations the analogue acts as an antagonist. Glycine binding is neither inhibited by glutamate nor by competitive inhibitors of glutamate, and does not bind at the same site as glutamate on the NMDA receptor. There is a second additional glycine binding site at the NMDA receptor. Thus, the ligand-gated ion channel of the NMDA receptor is under the control of at least two different allosteric sites. The disclosed compounds can be sites capable of binding or associating with the glycine binding site of the NMDA receptor. In some embodiments, the disclosed compounds have ten or more times the activity of the existing NMDA receptor glycine site partial agonist.

Compounds of the present disclosure may exhibit high therapeutic indices. Therapeutic index, as used herein, refers to the ratio of the dose causing toxicity in 50% of the population (ie, TD50) to the minimum effective dose in 50% of the population (ie, ED50). Thus, the therapeutic index _{= (TD 50) :( ED 50} ). In some embodiments, the disclosed compounds have a therapeutic index of at least about 10: 1, at least about 50: 1, at least about 100: 1, at least about 200: 1, at least about 500: 1, or at least about 1000: It is 1.

Compositions In another aspect, provided are formulations and compositions comprising a compound of the present disclosure and, optionally, a pharmaceutically acceptable excipient. In some embodiments, the formulation comprises a racemic mixture of one or more of the disclosed compounds.

  The formulations may be prepared in any of a variety of forms for use. Without limitation, and by way of example, the compounds are suitable for oral administration, subcutaneous injection or other methods for administering active agents to patients in need thereof as known in the pharmaceutical art It can be prepared into a formulation. For example, the pharmaceutical compositions of the present disclosure may be suitable for delivery to the eye. A related method may comprise administering a pharmaceutically effective amount of the disclosed compound or a pharmaceutical composition comprising the disclosed compound to the patient in need thereof, such as the eye of the patient, wherein administration is Local, subconjunctival, subretinal, intravitreous, retrobulbar, periocular, anterior chamber and / or systemic.

  The amount of disclosed compounds described herein in the formulation may vary depending on factors such as the individual's condition, age, sex and weight. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, or multiple divided doses may be administered over time, or doses may be increased or decreased depending on the urgency of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unit doses to the mammalian subject to be treated, each unit having a predetermined amount calculated to achieve the desired therapeutic effect. The active compound of the present invention is contained together with the required pharmaceutical carrier.

  The specification of the dosage unit form is inherent in the art of the synthesis of such active compounds to treat (a) the unique characteristics of the compound selected and the particular therapeutic effect to be achieved, and (b) the sensitivity of the individual. Depends on the limitations that you have, and it depends directly on this.

  Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to increase drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Sustained absorption can be brought about by including in the injectable composition an agent which delays absorption, for example, monostearate salts and gelatin.

  The compounds can be administered in sustained release formulations, such as compositions comprising sustained release polymers. The compounds can be prepared with carriers that protect the compound from immediate release, such as sustained release formulations including implants and microcapsule delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic acid, polyglycolic acid copolymers (PLG), and the like. Many methods for the preparation of such formulations are generally known to those skilled in the art.

  A sterile injectable solution can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum drying and lyophilization, in which the active ingredient and any additional powder of the desired ingredient are obtained from the solution which has previously been sterile filtered. .

  According to an alternative aspect, the compound may be formulated with one or more additional compounds that enhance the solubility of the compound.

Methods Administration of a therapeutically effective amount or a therapeutically effective dose of a compound described herein provides a method of treating a condition in a patient in need thereof. In some embodiments, the condition may be a psychiatric condition. For example, mental disorders can be treated. In another aspect, nervous system conditions may be treated. For example, conditions that develop in the central nervous system, peripheral nervous system and / or eye may be treated. In some embodiments, neurodegenerative diseases can be treated.

  In some embodiments, the method comprises autism, anxiety, depression, bipolar disorder, attention deficit disorder, attention deficit hyperactivity disorder (ADHD), schizophrenia, psychotic disorder, psychotic symptoms, social Withdrawal, obsessive compulsive disorder (OCD), phobia, post-traumatic stress syndrome, behavioral disorder, impulse control disorder, substance abuse disorder (eg withdrawal symptoms, opiate addiction, nicotine addiction and ethanol addiction), sleep disorder, memory Disorders (eg, loss, loss or diminished ability to remember newly), learning disorders, urinary incontinence, multiple system atrophy, progressive supranuclear palsy, Friedrich's ataxia, Down's syndrome, fragile X syndrome, nodular sclerosis Disease, olive bridge cerebellar atrophy, cerebral palsy, drug-induced optic neuritis, ischemic retinopathy, diabetic retinopathy, glaucoma, dementia, AIDS dementia, Alzheimer's disease, Huntington's chorea, convulsions Myoclonus, muscle spasm, sputum spat, Tourette's syndrome, epilepsy, cerebral ischemia, stroke, brain tumor, traumatic brain injury, cardiac arrest, myelopathy, spinal cord injury, peripheral neuropathy, acute neuropathic pain and chronic neurogenesis Including administering a compound to treat a patient suffering from sexual pain.

  In some embodiments, age-related memory impairment, schizophrenia, special learning disorders, seizures, seizures after stroke, cerebral ischemia, hypoglycemia, cardiac arrest, epilepsy, Lewy body dementia, migraine headaches, AIDS Methods are provided for treating dementia, Huntington's chorea, Parkinson's disease, early Alzheimer's disease and Alzheimer's disease.

  In certain embodiments, methods of treating schizophrenia are provided. For example, using the methods and compositions described herein, paranoid schizophrenia, disorganized schizophrenia (ie, catastrophic schizophrenia), stress-type schizophrenia, non-differentiated schizophrenia, Residual schizophrenia, post-schizophrenia depression and simple schizophrenia may be treated. The compositions described herein may also be used to treat psychotic disorders such as schizoaffective disorder, delusional disorder, short term psychotic disorder failure, shared psychotic disorder with delusions or hallucinations, etc. It can.

  A hallmark of delusional schizophrenia may be that although delusions or hallucinations are present, thought disorders, disorganized behavior or apathy are not observed. The delusion may be a paranoid and / or a paranoid, but in addition to these, there may be other themes such as jealousy, religiousness or somatization. A hallmark of disorganizing schizophrenia may be that both thinking disorder and apathy are seen. Stress-type schizophrenia may be that the patient is almost immobile or shows excited purposeless movements. Symptoms may include tonic stupor and waxy syndrome. Indistinguishable schizophrenia is characterized by psychotic symptoms but may not meet the criteria of delusional, disorganized or tense. The hallmark of residual schizophrenia may be that only positive symptoms are seen. Post-schizophrenic depression may be characterized by the effects of schizophrenia resulting in depressive episodes and still have some low-level symptoms of schizophrenia. Simple schizophrenia may be characterized as a mild progression of prominent negative symptoms without a history of psychotic episodes.

  In some embodiments, provided are methods of treating psychotic conditions that may be present in other mental disorders including, but not limited to, bipolar disorder, borderline personality disorder, drug addiction and drug-induced psychosis. In certain embodiments, provided are methods of treating delusions (eg, “non-quiet”) delusions that may be seen, for example, in delusional disorders.

  Various embodiments provide methods of treating social withdrawal found in conditions including, but not limited to, social anxiety disorders, avoidance personality disorders and schizotypal personality disorders.

  In some embodiments, the disclosure provides a method for treating a neurodevelopmental disorder associated with synaptic dysfunction in a patient in need thereof, wherein the method generally comprises a therapeutically effective amount. Administering a pharmaceutical composition comprising the disclosed compound or the disclosed compound to a patient. In certain embodiments, the neurodevelopmental disorder associated with synaptic dysfunction is cerebral atrophic hyperammonemia, MECP2 duplication syndrome (eg, MECP2 disorder), CDKL5 syndrome, fragile X syndrome (eg, FMR1 disorder), tuberous sclerosis Disorders (eg, TSC1 disorder and / or TSC2 disorder), neurofibromatosis (eg, NF1 disorder), Angelman's syndrome (eg, UBE3A disorder), PTEN hamartoma tumor syndrome, Phelan-McDermid syndrome (eg, SHANK3 disorder) Or Rett syndrome, also known as infantile spasms. In certain embodiments, neurodevelopmental disorders may be caused by mutations of neurolysine (eg, NLGN3 and / or NLGN2 disorders) and / or neurexins (eg, NRXN1 disorders).

  In some embodiments, methods of treating neuropathic pain are provided. Neuropathic pain may be acute or chronic. In some cases, neuropathic pain may be herpes, HIV, traumatic nerve injury, stroke, post ischemia, chronic back pain, postherpetic neuralgia, fibromyalgia, reflex sympathetic dystrophy, complex regional pain It may be associated with pathologies such as syndromes, spinal cord injury, sciatica, phantom pain, diabetic neuropathy such as diabetic peripheral neuropathy (DPN), and neuropathic pain with cancer chemotherapy. Also provided are methods of enhancing pain relief and methods of providing pain relief to a patient.

  In various embodiments, the methods of the present disclosure include treating an autism and / or autism spectrum disorder in a patient in need thereof comprising administering to the patient an effective amount of the disclosed compound. Including how to do it. In some embodiments, provided is a method of reducing autism symptoms in a patient in need thereof, wherein the method comprises administering to the patient an effective amount of the disclosed compound. . For example, administration of the compound may cause symptoms of autism, such as not being in line of sight, being unable to keep in touch, being alert, poor emotional, hyperactive, abnormally hypersensitive to sounds, inappropriate conversation, sleep The frequency of occurrence of one or more symptoms such as disability and adherence may be reduced. Such a reduction in frequency of occurrence may be measured relative to the frequency of occurrence in untreated individuals.

  Also provided herein are methods of modulating autism target gene expression in cells, comprising contacting the cells with an effective amount of a compound described herein. Autism gene expression can be selected from, for example, ABAT, APOE, CHRNA4, GABRA5, GFAP, GRIN2A, PDYN and PENK. In certain embodiments, provided is a method of modulating synaptic plasticity in a patient suffering from a disorder associated with synaptic plasticity comprising administering to the patient an effective amount of a compound.

In some embodiments, provided is a method of treating Alzheimer's disease in a patient in need thereof, comprising administering the compound, or treating memory loss that occurs with, for example, early Alzheimer's disease. As used herein, in vitro or in vivo (eg, intracellular) Alzheimer's disease amyloid protein (eg, β-amyloid peptide, eg, isoform Aβ), comprising contacting the Alzheimer's disease amyloid protein with an effective amount of a disclosed compound A method of adjusting _1-4 ) is also provided. For example, in some embodiments, compounds can block the ability of such amyloid proteins to inhibit long-term potentiation in hippocampal slices and neuronal cell death by apoptosis. In some embodiments, the disclosed compounds may provide neuroprotective properties to Alzheimer's disease patients in need thereof, such as therapeutic effects on neuronal cell death due to late Alzheimer's disease.

  In certain embodiments, the disclosed methods include stroke, amyotrophic lateral sclerosis (ALS or Lugueric disease), multiple sclerosis, traumatic brain injury, Alzheimer's disease, dementia and / or Parkinson's disease, etc. Treating psychosis or pseudobulb emotion ("PBA") induced by another medical condition. Such methods, as well as other methods of the present disclosure, comprise administering a pharmaceutically effective amount of the disclosed compound to a patient in need thereof.

  In various embodiments, provided is a method of treating depression comprising administering a compound described herein. In some embodiments, the treatment may relieve depression or symptoms of depression without affecting behavioral or motor coordination and without inducing or promoting seizure activity. Exemplary conditions of depression expected to be treated according to this aspect include, but are not limited to, major depressive disorder, dysthymic disorder, psychotic depression, postpartum depression, premenstrual syndrome Premenstrual dysphoric disorder, seasonal affective disorder (SAD), bipolar disorder (or depressive disorder), mood disorder, chronic medical condition such as cancer or chronic pain, chemotherapy, chronic stress etc. Depression and post-traumatic stress disorder. In addition, patients suffering from any form of depression are often anxious. Various symptoms caused by anxiety include especially fear, panic, heart palpitations, shortness of breath, fatigue, nausea and headache. Anxiety or any of its symptoms may be treated by administering the compounds described herein.

  As used herein, a treatment resistant patient, eg, suffering from a mental or central nervous system condition that has not responded to and / or has not responded to appropriate treatment with at least one or at least two compounds or agents. Also provided are methods of treating the condition of a subject. For example, as used herein, depression of a treatment resistant patient, comprising: a) optionally confirming that the patient is treatment resistant; and b) administering an effective amount of a compound to said patient. Methods of treatment are provided.

  In some embodiments, the compounds described herein may be used for acute treatment of patients. For example, the compound is administered to a patient to treat a particular episode (eg, a severe episode) of a condition described herein.

  Also included herein are combination therapies comprising the disclosed compounds in combination with one or more other active agents. For example, the disclosed compounds can be combined with one or more antidepressants, such as tricyclic antidepressants, MAO-I, SSRIs, dual uptake inhibitors, triple uptake inhibitors and the like and / or anxiolytics. Exemplary drugs which may be used in combination with the compound include anafranil, adapine, aventil, elavir, norpramine, pamelol, part-furan, senequan, sulmontyl, tofranil, bibactil, parnate, nadir, marplan, lexapro, lobox, paxil, prozac, Zoloft, Hulvatrin, effector, Remelon, Cymbalta, Desierel (Trazodone) and Lugiomeal. In another example, the compound may be combined with an antipsychotic drug. Non-limiting examples of antipsychotic agents include butyrophenone, phenothiazine, thioxanthene, clozapine, olanzapine, risperidone, quetiapine, ziprasidone, amisulpride, asenapine, paliperidone, iloperidone, zotepine, sertindole, lurasidone and aripiprazole. It should be understood that the combination of a compound and one or more of the above therapeutic agents may be used in the treatment of any suitable condition, and is not limited to use as an antidepressant or an antipsychotic.

  The following examples are provided for the purpose of illustration only and are not intended to limit the scope of the present disclosure.

The following abbreviations may be used herein and they have the following definition: Ac is acetyl (-C (O) CH ₃ ), and AIDS is acquired immunodeficiency syndrome , Boc and BOC are tert-butoxycarbonyl, Boc ₂ O is di-tert-butyl dicarbonate, B n is benzyl, BOM-Cl is benzyloxymethyl chloride, and CAN is Ceric ammonium nitrate, Cbz is carboxybenzyl, DCM is dichloromethane, DIAD is diisopropylazodicarboxylate, DIPEA is N, N-diisopropylethylamine, DMAP is 4-dimethyl Aminopyridine, DMF is N, N-dimethylformamide, DM SO is dimethyl sulfoxide, EDC and EDCI are 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, ESI is electrospray ionization, EtOAc is ethyl acetate, Gly Is glycine, h is time, HATU is 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate , HIV is human immunodeficiency virus, HOBt is hydroxybenzotriazole, HPLC is high performance liquid chromatography, LCMS is liquid chromatography / mass spectrometry, LDA is lithium diisopropylamide Yes, LiHMDS is a lithium hexamethyl disila MTBE is methyl tert-butyl ether, NMDAR is N-methyl-d-aspartate acceptor, NMP is N-methyl-2-pyrrolidone, NMR is nuclear magnetic resonance Pd / C is palladium on carbon, PMB is paramethoxybenzyl, RT is room temperature (eg, about 20 ° C. to about 25 ° C.), TBS and TBDMS are tert-butyldimethylsilyl , TEA is triethylamine, TLC is thin-layer chromatography, TFA is trifluoroacetic acid, THF is tetrahydrofuran, TMS is trimethylsilyl, TMSCN is trimethylsilyl cyanide Yes, TPP is triphenyl phosphine.

Example 1-Synthesis of Compound A and Compound A Maleate
Synthesis of (3R, 7aS) -3- (trichloromethyl) tetrahydro-1H, 3H-pyrrolo [1,2-c] oxazol-1-one (1):
To a suspension of L-proline (2.0 kg, 0.017 mol) in chloroform (50 L) at room temperature chloral hydrate (5.7 kg, 0.034 mol) was added. The reaction mixture was heated to 60 ° C. under a reverse Dean-Stark apparatus and the resulting water collected. After 16 hours, the volatiles were evaporated under reduced pressure. The crude solid was washed with cold ethanol, filtered and dried to give compound 1 (2.2 kg, 57%) as a white solid. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 5.82 (s, 1 H), 4.11 to 4.08 (m, 1 H), 3.33 to 3.27 (m, 2 H), 3 .19-3.14 (m, 1 H), 2.15-2.10 (m, 1 H), 1.96-1.91 (m, 1 H), 1.80-1.74 (m, 1 H) , 1.65-1.58 (m, 1 H).

Synthesis of (3R, 7aR) -7a-((benzyloxy) methyl) -3- (trichloromethyl) tetrahydro-1H, 3H-pyrrolo [1,2-c] oxazol-1-one (2):
N-BuLi (1.6 M in hexane) (958.5 mL, 1.533 mol) was added dropwise to a solution of diisopropylamine (221.2 mL, 1.533 mol) in THF (870 mL) at -78 C under a nitrogen atmosphere. After the addition was complete, the temperature of the reaction mixture was raised to -20.degree. C. and stirred for 1 hour. It was again cooled to −78 ° C., Compound 1 (250 g, 1.022 mol) in THF (1 L) was added and stirred for 30 minutes. Then, benzyl chloromethyl ether (208 mL, 1.329 mol) was added dropwise and stirring was continued for 1 hour. After the starting material had been consumed (by TLC), the reaction mixture was quenched with ice cold water (100 mL) and extracted with Et ₂ O (2 × 100 mL). The combined organic layers are washed with brine (100 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound which is purified by column chromatography eluting with 10% EtOAc / n-hexane Then, compound 2 (220 g, crude) was obtained as a brownish viscous syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 7.42 to 7.26 (m, 5 H), 5.60 (s, 1 H), 4.59 to 4.53 (d, 2 H), 3 .67-3.61 (m, 2H), 3.37-3.33 (m, 1H), 3.31-3.10 (m, 1H), 2.12-1.99 (m, 2H) , 1.87-1.75 (m, 2H); LCMS (ESI): m / z 363.9 [M ⁺ +1].

Synthesis of methyl (R) -2-((benzyloxy) methyl) pyrrolidine-2-carboxylate hydrochloride (3):
To a solution of compound 2 (400 g, 1.096 mol) in methanol (1 L) at room temperature was added 2N HCl in MeOH (1.64 L, 3.29 mol) and stirred at 80 ° C. for 16 hours. After the starting material was consumed (by TLC), the volatiles were evaporated under reduced pressure. The crude product obtained was washed with hexane (3 × 750 mL) and dried under reduced pressure to give compound 3 (358 g, crude) as a reddish viscous syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 10.40 (br s, 1 H), 7.40 to 7.21 (m, 5 H), 4.64 to 4.50 (d, 2 H), 4.49-4.3 (d, 2 H), 3.76 (s, 3 H), 3.33-3.22 (m, 2 H), 2.22-2.15 (s, 1 H), 02-1.9 (m, 2H), 1.95 to 1.83 (m, 1H).

Synthesis of 1- (tert-Butyl) 2-methyl (R) -2-((benzyloxy) methyl) pyrrolidine-1,2-dicarboxylate (4):
To a stirred suspension of compound 3 (313 g, crude 1.096 mol) in DCM (2.19 L) at 0 ° C. under a nitrogen atmosphere Et ₃ N (458.4 mL, 3.288 mol) is added dropwise and stirred for 10 minutes did. Then Boc anhydride (358.4 g, 1.644 mol) was added dropwise at 0 ° C. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. After starting material was consumed (by TLC), the reaction was diluted with water (2 × 1 L) and extracted with CH ₂ Cl ₂ (2 × 500 mL). The combined organic layers were washed with 10% citric acid (pH ̃7) and brine solution (1 L). The organic layer is dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound which is purified by column chromatography eluting with 20% EtOAc / n-hexane to give compound 4 (215 g, 56%) Was obtained as a colorless viscous syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 7.37-7.26 (m, 5 H), 4.54-4.48 (m, 2 H), 4.03-3.87 (dd, 1 H), 3.69-3.67 (d, 1 H), 3.62 (s, 3 H), 3.53-3.47 (m, 1 H), 3.33-3.30 (m, 1 H) , 2.27-2.20 (m, 1 H), 2.03 to 1.89 (m, 2 H), 1.88 to 1.79 (m, 1 H), 1.46 to 1.24 (2 s, 2 s) 9 H); LCMS (ESI): m / z 250.1 [(M ⁺ +1) -Boc].

Synthesis of (R) -2-((benzyloxy) methyl) -1- (tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid (5):
NaOH (73.9 g, 1.848 mol) was added to a solution of compound 4 (215 g, 0.616 mol) in MeOH: THF: H ₂ O (3 L, 5: 5: 3) and stirred at room temperature for 10 minutes. The reaction mixture was heated to 80 ° C. and stirred for 16 hours. After the starting material was consumed (by TLC), the reaction mixture was brought to room temperature and the volatiles were evaporated. The crude material was diluted with water (1 L) and extracted with Et ₂ O (2 × 500 mL). The separated aqueous layer was acidified with 2N HCl solution (pH ̃3) and extracted with DCM (2 × 750 mL). The combined organic layers were washed with brine solution, dried over Na ₂ SO ₄ and concentrated to give compound 5 (170 g, 82.4%) as an off-white solid. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 12.66 (br s, 1 H), 7.36 to 7.26 (m, 5 H), 4.54 to 4.47 (m, 2 H), 4.05 to 3.90 (dd, 1 H), 3.88 to 3.63 (d, 1 H), 3.63 to 3.44 (m, 1 H), 3.34 to 3.27 (m, 1 H) ), 2.27-2.01 (m, 1 H), 2.02-1.8 (m, 2 H), 1.79-1.76 (m, 1 H), 1.17-1.14 (2 s) , 9H); LCMS (ESI): m / z 235.1 [(M ⁺ +1) -Boc].

Synthesis of (R) -1- (tert-butoxycarbonyl) -2- (hydroxymethyl) pyrrolidine-2-carboxylic acid (6):
To a stirred solution of compound 5 (170 g, 0.507 mol) in CH ₃ OH (1 L) at room temperature was added 50% wet 10% Pd—C (68 g) and stirred under H ₂ atmosphere for 16 hours. After starting material was consumed (by TLC), the reaction mixture was filtered through a pad of celite and the pad was washed with CH ₃ OH (1 L). The resulting filtrate was concentrated under reduced pressure to give compound 6 (110 g, 88%) as a white solid. ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 12.66 (br s, 1 H), 3.96 to 3.83 (dd, 1 H), 3.63 to 3.60 (m, 1 H), 3.49-3.46 (m, 1 H), 3.34-3.25 (m, 2 H), 2.30-2.15 (m, 1 H), 1.95-1.72 (m, 3 H) LCS (ESI): m / z 244 [M ⁺ -1]; Chiral HPLC: 95. 88%.), 1.38-1.33 (2s, 9H);

tert-Butyl (R) -2-(((2S, 3R) -1,3-bis (benzyloxy) -1-oxobutan-2-yl) carbamoyl) -2- (hydroxymethyl) pyrrolidine-1-carboxylate Synthesis of (7):
A stirred solution of compound 6 (110 g, 0.448 mol) in DCM (20 mL) cooled to 0 ° C. under N ₂ atmosphere with diisopropylethylamine (206 mL, 1.12 mol), intermediate D1 (150 g, 0.448 mol) and HATU (204 g, 0.537 mol) was added. The reaction mixture was allowed to reach room temperature and stirred for 4 hours. After starting material was consumed (by TLC), the reaction mixture was diluted with DCM (1 L) and washed with water (2 × 500 mL), 10% citric acid solution (500 mL) and brine solution (500 mL). The organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound which is purified by column chromatography eluting with 30% EtOAc / n-hexane to give compound 7 (143 g, 60% ) Was obtained as a colorless viscous syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 8.20-8.12 (d, 1 H), 7.29-7.18 (m, 10 H), 5.83-5.59 (m, 1H), 5.08 (s, 2H), 4.50 to 4.44 (m, 2H), 4.30 to 4.26 (m, 1H), 4.08 to 4.00 (m, 2H) , 3.42 to 3.40 (m, 2 H), 3.39 to 3.29 (m, 1 H), 2.19 to 2.08 (m, 1 H), 1.96 to 1.87 (m, 1) 1H), 1.68-1.63 (m, 2H), 1.23-1.15 (2s, 9H), 1.4-1.13 (d, 3H); LCMS (m / z): 525 ^{.2 [M + -1]; HPLC} : 76.2%; ChiralHPLC: 69.47%.

tert-Butyl (R) -2-((2S, 3R) -1,3-bis (benzyloxy) -1-oxobutan-2-yl) -1-oxo-2,5-diazaspiro [3.4] octane Synthesis of -5-carboxylate (8):
To a solution of triphenylphosphine (161.4 g, 0.612 mol) in THF (430 mL) was added dropwise DIAD (123.8 g, 0.612 mol) at room temperature under a nitrogen atmosphere and stirred for 15 minutes. A solution of compound 7 (215 g, 0.408 mol) in THF (860 mL) was added dropwise thereto, and the mixture was stirred at room temperature for 4 hours. After the starting material was consumed (by TLC), the volatiles were evaporated under reduced pressure. The crude material obtained was purified by silica gel column chromatography eluting with 20% EtOAc / hexane to give compound 8 (180 g, mixture with DIAD by-product) as a yellow syrup. ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 7.31 to 7.18 (m, 10 H), 5.18 to 5.10 (m, 2 H), 4.61 to 4.54 (m, 10) 2H), 4.27-4.18 (m, 2H), 3.78-3.77 (d, 1 H), 3.45-3.43 (d, 1 H), 3.35-3. 31 (m) d, 1H), 3.27-3.23 (m, 1H), 2.03-1.98 (m, 2H), 1.78-1.76 (m, 2H), 1.39-1. 31 (2s, 9H), 1.23-1.22 (d, 3H) (peak of DIAD by-product not captured); LCMS (ESI): m / z 509.4 [M ⁺ +1].

(2S, 3R) -2-((R) -5- (tert-butoxycarbonyl) -1-oxo-2,5-diazaspiro [3.4] octan-2-yl) -3-hydroxybutanoic acid (9 Synthesis of):
A solution of compound 8 (85 g, 0.167 mol) in methanol (850 mL) was degassed under a N ₂ atmosphere. Then 50% wet 10% Pd-C (40 g) was added at room temperature and stirred for 24 h under H ₂ atmosphere. After starting material was consumed (by TLC), the reaction mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The crude solid obtained was diluted with Et ₂ O (1 L) and stirred vigorously at room temperature for 1 h. The resulting solid was filtered off and dried to give compound 9 (75 g, 68%) as a white solid. ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 12.86 (br s, 1 H), 5.24 (br s 1 H), 4.06-4.00 (m, 2 H), 3.88- 3.82 (m, 1 H), 3.51-3. 50 (m, 1 H), 3.43-3.24 (m, 1 H), 3.31-3. 23 (m, 1 H), 15-2.09 (m, 2H), 1.82-1.78 (m, 2H), 1.39-1.31 (2s, 9H), 1.10-1.08 (d, 3H); LCMS (m / z): 329.1 [M ⁺ +1]; HPLC: 93.97%.

tert-Butyl (R) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl) -1-oxo-2,5-diazaspiro [3.4] octane-5 -Synthesis of Carboxylate (A4):
A stirred solution of compound 9 (150 g, 0.457 mol) in CH ₂ Cl ₂ ( ₂ L) at 0 ° C. in diisopropylethylamine (176.85 g, 1.37 mol), NH ₄ Cl (48.8 g, 0.914 mol) and HATU (208.3 g 0.548 mol) was added under a nitrogen atmosphere. The reaction mixture was allowed to reach room temperature and stirred for 12 hours. After starting material was consumed (by TLC), the reaction mixture was diluted with water (2 × 750 mL) and extracted with CH ₂ Cl ₂ (2 × 1 L). The combined organic layers were washed with 2N HCl solution and saturated sodium chloride solution (750 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude compound was purified by column chromatography and pure compound was eluted with 3% MeOH / CH ₂ Cl ₂ . The resulting material was diluted with DCM (2 L) and washed with saturated citric acid (5 × 500 mL), saturated bicarbonate wash followed by brine wash and dried over Na ₂ SO ₄ . The material was triturated with ether (2 × 500 mL) to give compound A4 (80 g, 53%) as a white solid. ¹ H-NMR: (400 MHz, D ₂ O): δ 4.36 to 4.21 (m, 2 H), 4.03 to 3.98 (m, 1 H), 3.76 to 3.67 (m, 1 H) ), 3.56-. 3.37 (m, 2H), 2.32-2.23 (m, 2H), 1.97-1.93 (m, 2H), 1.49-1.47 (2s, 9H), 1. 33-1.32 (d, 3H); LCMS (ESI): m / z328.3 [M + +1]; HPLC: 99.30%; chiral HPLC: 98.97%; SOR (c = 1, CH 3 OH): −1.08.

Synthesis of (2S, 3R) -3-hydroxy-2-((R) -1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (10):
To a stirred solution of compound A4 (10 g, 0.030 mol) in CH ₂ Cl ₂ (50 mL) at 0 ° C. under nitrogen atmosphere was added TFA (24 mL, 0.3058 mol). The reaction mixture was allowed to reach room temperature and stirred for 4 hours. After the starting material was consumed (by TLC), the volatiles were evaporated under reduced pressure. The resulting thick syrup material was washed with ether (2 × 100 mL) and hexane (1 × 100 mL) and dried under vacuum to give compound 10 (3.5 g, TFA salt) as a thick syrup , Which was used in the next step. The crude RM was carried forward without purification. LCMS (ESI): m / z 228.1 [M ⁺ +1].

Synthesis of (2S, 3R) -3-hydroxy-2-((R) -5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (Compound A):
To a stirred solution of compound 10 (10 g, 0.028 mol TFA salt) in methanol (50 mL) isobutyraldehyde (10 mL, 0.115 mmol) followed by AcOH (12 mL) at room temperature under nitrogen and stirred for 10 minutes did. Then NaCNBH ₃ (7.2 g, 11.5 mmol) was added in small portions and stirring was continued for 16 hours. After the starting material was consumed (by TLC), the reaction mixture was diluted with CH ₂ Cl ₂ (10 mL) and concentrated under reduced pressure. The same reaction was performed in batches of 5 g. Both lots are combined and the resulting crude material is purified by silica gel column chromatography eluting with 4% MeOH / DCM to give (2S, 3R) -3-hydroxy-2-((R) -5-isobutyl- A mixture of 1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide was obtained. The resulting mixture is further subjected to column chromatography to give (2S, 3R) -3-hydroxy-2-((R) -5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octane-2 -Yl) Butanamide (8 g) is obtained as a thick syrup. ¹ H-NMR: (500 MHz, CD ₃ OD): δ 4.16 to 4.12 (m, 1 H), 4.09 (d, J = 7.0 Hz, 1 H), 3.60 (d, J = 6) .0 Hz, 1 H), 3.54 (d, J = 6.0 Hz, 1 H), 2.97-2.93 (m, 1 H), 2.75 (q, J = 8.0 Hz, 1 H), 2 .47-2.45 (m, 2 H), 2.22-2.18 (m, 1 H), 2.16-2.10 (m, 1 H), 1.92-1.83 (m, 2 H) , 1.76-1.73 (m, 1 H), 1.24 (d, J = 6.0 Hz, 3 H), 0.92-0.89 (m, 6 H); LCMS (ESI): m / z 284 ^{.1 [M + +1]; HPLC} : 97.65%; chiral _{HPLC: 98.25%; SOR (c} = 1, CH 3 OH): 1.6.

(4R) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl) -5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octane- Synthesis of 5-ium (Z) -3-Carboxy Acrylate (Compound A Maleate):
(2S, 3R) -3-Hydroxy-2-((R) -5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (400 mg, 1.41 mmol) To the solution was added maleic acid (148 mg, 1.27 mmol) at room temperature and stirred for 16 hours. After the starting material was consumed (by TLC), the water was removed under reduced pressure. The crude material obtained is triturated with pentane and hexane, dried under vacuum and (4R) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl) 5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octan-5-ium (Z) -3-carboxyacrylate (530 mg, 94%) was obtained as a white solid. ¹ H NMR (500 MHz, CD ₃ OD) δ 6.29 (s, 2 H), 4.21-4.12 (m, 2 H), 3.91-3. 85 (m, 1 H), 3.83-3 .79 (m, 1 H), 3.61-3.51 (m, 1 H), 3.29-3.19 (m, 1 H), 3.04 (dd, J = 9.0, 12.5 Hz, 1 H), 2.90 (dd, J = 5.8, 12.2 Hz, 1 H), 2.52-2.43 (m, 1 H), 2.40-2.31 (m, 1 H), 2. H. 25-1.97 (m, 3 H), 1.28 (d, J = 5.8 Hz, 3 H), 1.04 (dd, J = 6.4, 9.9 Hz, 6 H); LCMS (ESI): m / z 284.3 [M ⁺ + 1-maleic acid]; HPLC: 96.83%; melting point: 113.4 ° C-117.5 ° C.

Preparation of Int-D:
Synthesis of (tert-butoxycarbonyl) -L-threonine (A1):
To a solution of SM-2 (50 g, 0.420 mol) in 1,4-dioxane: water (500 mL, 1: 1) NaHCO ₃ (133 g, 1.255 mol) was added portionwise at room temperature and stirred for 15 minutes. Then Boc anhydride (144 mL, 0.629 mol) was added dropwise to the reaction mixture and stirring was continued at room temperature for 16 hours. After starting material was consumed (by TLC), the reaction mixture was concentrated under reduced pressure and the residue obtained was diluted with water (200 mL) and acidified by using 1N HCl (pH ̃2). The aqueous layer was extracted with EtOAc (2 × 300 mL). The combined organic layers were washed with brine (1 × 200 mL), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give A1 (80 g, 87%) as a colorless syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 12.5 (br s, 1 H), 6.30 (d, J = 8.5 Hz, 1 H), 4.50 (br s, 1 H), 4 .05-4.02 (m, 1 H), 3.88-3.86 (m, 1 H), 1.39 (s, 9 H), 1.08 (d, J = 6.0 Hz, 3 H);
LCMS (m / z): 218.1 [M ⁺ -1]

Synthesis of O-benzyl-N- (tert-butoxycarbonyl) -L-threonine (B1):
DMF (800 mL) compounds in A1 (80g, 0.365mol) stirring solution of 60% -20 ° C. in a _{N 2} atmosphere NaH (22g, 0.913mol) was added portionwise and stirred for 2 hours. To this was added benzyl bromide (52 mL, 0.438 mol) dropwise and the reaction mixture was stirred at 0 ° C. for 4 hours. After the starting material had been consumed (by TLC), the reaction mixture was quenched with ice cold water and extracted with diethyl ether (2 × 500 mL). The aqueous layer was acidified with 1N HCl (pH ̃2). The aqueous layer was extracted with EtOAc (2 × 1 L). The separated organic layer was washed with brine solution, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give compound B1 (84 g, crude) as a thick syrup.
¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 12.64 (br s, 1 H), 7.34-7.25 (m, 5 H), 6.46 (d, J = 8.5 Hz, 1 H) ), 4.53 (d, J = 11.5 Hz, 1 H), 4.39 (d, J = 12.0 Hz, 1 H), 4.00-3.98 (m, 2 H), 1.39 (s) , 9H), 1.15 (d, J = 6.0 Hz, 3 H).

Synthesis of benzyl O-benzyl-N- (tert-butoxycarbonyl) -L-threonic acid (C1):
To a stirred solution of compound B1 (78 g, 0.252 mol) in DMF (780 mL) was added K ₂ CO ₃ (87 g, 0.631 mol) at room temperature under N ₂ atmosphere and stirred for 30 minutes. To this was added benzyl bromide (45 mL, 0.378 mol) dropwise at room temperature and stirred for 16 hours. The reaction mixture was quenched with water (2 L) and extracted with diethyl ether (2 × 1 L). The separated organic layer was washed with brine, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography eluting with 10% EtOAc / n-hexane to give compound C1 (68 g, 68%) as a yellow syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 7.37-7.18 (m, ¹⁰ H), 6.81 (d, J = 9.0 Hz, 1 H), 5.08 (s, 2 H) , 4.49 (d, J = 12.0 Hz, 1 H), 4.32 (d, J = 1 2.0 Hz, 1 H), 4.25 to 4.22 (m, 1 H), 4.01-3. 98 (m, 1 H), 1. 38 (s, 9 H), 1. 15 (d, J = 6.0 Hz, 3 H); mass (ESI): m / z 399.4 [M ⁺ ].

Synthesis of benzyl O-benzyl-L-threonine hydrochloride (D1):
To a solution of compound C1 (68 g, 0.170 mol) in diethyl ether (500 mL) was added 4 N HCl (130 mL, 0.511 mol) in 1,4-dioxane and stirred at room temperature for 16 hours. After the starting material was consumed (by TLC), the reaction mixture was concentrated under reduced pressure. The crude material was dissolved in diethyl ether (1 L) and stirred vigorously at room temperature for 1 hour. The resulting solid was filtered off and dried under reduced pressure to give compound D1 (50 g, 87%) as a white solid (HCl salt). ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 8.59 (s, 2 H), 7. 50-7. 25 (m, 10 H), 5.23 (d, J = 12.5 Hz, 1 H) , 5.16 (d, J = 12.5 Hz, 1H), 4.54 (d, J = 12.0 Hz, 1 H), 4.36 (d, J = 12.0 Hz, 1 H), 4.12- 4.09 (m, 1 H), 4.09-3.99 (m, 1 H), 1. 29 (d, J = 6.5 Hz, 3 H); Mass (ESI): m / z 336.4 [M ⁺ +1].

Example 2-Synthesis of Compound B and Compound B Maleate
Synthesis of (3S, 7aR) -3- (trichloromethyl) tetrahydro-1H, 3H-pyrrolo [1,2-c] oxazol-1-one (1):
To a suspension of D-proline (3 kg, 26.057 mol) in chloroform (60 L) was added chloral hydrate (8.62 kg, 52. 115 mol) at room temperature. The reaction mixture was heated to 80 ° C. under a reverse Dean-Stark apparatus and the resulting water collected. After stirring for 24 hours, the reaction mixture was cooled to room temperature and brine solution (25 L) was added. The reaction mixture was stirred for 30 minutes and allowed to stand for 30 minutes. The separated organic layer was dried over Na ₂ SO ₄ , concentrated under reduced pressure and the volatiles evaporated under reduced pressure. The crude solid obtained was slurried with cold ethanol (5 L), filtered and dried to give compound 1 (4.7 kg, 74%) as an off-white solid. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 5.82 (s, 1 H), 4.11 to 4.09 (m, 1 H), 3.33 to 3.28 (m, 1 H), 3 .19-3.14 (m, 1 H), 2.17-2.10 (m, 1 H), 1.97-1.91 (m, 1 H), 1.80-1.74 (m, 1 H) , 1.65-1.58 (m, 1 H).

Synthesis of (3S, 7aS) -7a-((benzyloxy) methyl) -3- (trichloromethyl) tetrahydro-1H, 3H-pyrrolo [1,2-c] oxazol-1-one (2):
N-BuLi (1.6 M in hexane) (1.91 L, 3.067 mol) was added dropwise to a solution of diisopropylamine (442 mL, 3.067 mol) in THF (1 L) at -78 C under a nitrogen atmosphere. After the addition was complete, the temperature of the reaction mixture was raised to -20.degree. C. and stirred for 45 minutes. It was again cooled to −78 ° C., Compound 1 (500 g, 2.044 mol) in THF (2 L) was added dropwise and stirred for 45 minutes. Then benzyl chloromethyl ether (425 mL, 3.067 mol) was added dropwise and stirring was continued for 2 hours. After the starting material was consumed (by TLC), the reaction mixture was quenched with ice cold water (2 L) and extracted with Et ₂ O (2 × 1 L). The combined organic layers are washed with brine (3 L), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound which is purified by column chromatography eluting with 10% EtOAc / n-hexane Compound 2 (425 g, 57%) was obtained as a yellow liquid. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 7.35 to 7.26 (m, 5 H), 5.60 (s, 1 H), 4.59 to 4.53 (d, 2 H), 3 .67-3.62 (m, 2 H), 3.71-3. 31 (m, 1 H), 3.16-3.11 (m, 1 H), 2.12-1.99 (m, 2 H) , 1.86-1.72 (m, 2H); LCMS (ESI): m / z 363.8 [M ⁺ -1].

Synthesis of methyl (S) -2-((benzyloxy) methyl) pyrrolidine-2-carboxylate hydrochloride (3):
To a solution of compound 2 (1 kg, 2.742 mol) in methanol (2 L) was added 2 N HCl in methanol (4.1 L, 8.227 mol) at room temperature and stirred for 30 minutes. The reaction mixture was stirred at 60 ° C. for 16 hours. After the starting material was consumed (by TLC), the volatiles were evaporated under reduced pressure. The crude syrup obtained was diluted with DM water (3 L) and washed with EtOAc (2 × 1 L). The aqueous layer pH (8-9) was adjusted with NaHCO3 solution. The aqueous layer was extracted with 5% MeOH / DCM (2 × 2 L). The combined organic layers were washed with brine solution, dried over Na ₂ SO ₄ and concentrated under reduced pressure to give compound 3 (600 g, 88%) as a brown syrup. Another 1 kg batch was repeated to obtain 600 g of compound 3. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 10.54 (br s, 1 H), 9.44 (br s, 1 H), 7.40 to 7.30 (m, 5 H), 4.68 −4.62 (d, 1 H), 4.51 to 4.46 (d, 1 H), 4.00 to 3.90 (m, 2 H), 3.76 (s, 3 H), 3.33-3 16 (m, 2 H), 2.20-2.14 (s, 1 H), 2.01-1. 85 (m, 3 H).

Synthesis of 1- (tert-Butyl) 2-methyl (S) -2-((benzyloxy) methyl) pyrrolidine-1,2-dicarboxylate (4):
Et ₃ N (1.35 L, 9.638 mol) was added dropwise to a stirred suspension of compound 3 (1.2 kg, 4.819 mol) in DCM (6 L) at 0 ° C. under a nitrogen atmosphere and stirred for 20 minutes . Then Boc anhydride (1.65 L, 7.228 mol) was added dropwise at 0 ° C. The reaction mixture was allowed to reach room temperature and stirred for 8 hours. After starting material was consumed (by TLC), the reaction was diluted with DM water (3 L) and extracted with DCM (2 L). The combined organic layers were washed with 10% citric acid (2 L) solution and brine solution (2 L). The organic layer was dried over Na ₂ SO ₄ and concentrated under reduced pressure to give compound 4 (2 kg, crude) as a brown syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 7.37-7.28 (m, 5 H), 4.57-4. 50 (m, 2 H), 4.03-3.88 (dd, 1H), 3.71-3.69 (d, 1 H), 3.63 (s, 3 H), 3.52-3.47 (m, 1 H), 3.36-3.31 (m, 1 H) , 2.32-2.23 (m, 1 H), 2.05-1.88 (m, 2 H), 1.82-1.77 (m, 1 H), 1.38-1.26 (s, 1 2) 9 H); LCMS (ESI): m / z 350.2 [(M ⁺ +1)].

Synthesis of (S) -2-((benzyloxy) methyl) -1- (tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid (5):
NaOH (343.84 g, 8.595 mol) in H ₂ O (1.5 L) is added to a solution of compound 4 (1 kg, 2.865 mol) in MeOH: THF (3 L, 1: 1) at room temperature, 70 ° C. Stir for 6 hours. After the starting material was consumed (by TLC), the reaction mixture was allowed to reach room temperature and the volatiles were evaporated under reduced pressure. The crude material was diluted with water (5 L) and extracted with EtOAc (2 × 2 L). The separated aqueous layer was acidified with 6 N HCl solution (pH ̃2) and extracted with DCM (3 × 2 L). The combined organic layers were washed with brine solution, dried over Na ₂ SO ₄ and concentrated to give compound 5 (550 g, 58%) as a brown syrup. Another 1 kg batch was repeated to give 550 g of compound 5. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 12.68 (br s, 1 H), 7.36 to 7.28 (m, 5 H), 4.56 to 4.49 (m, 2 H), 4.07 to 3.90 (dd, 1 H), 3.66 to 3.64 (d, 1 H), 3.50 to 3.44 (m, 1 H), 2.29 to 2.20 (m, 1 H) ), 2.03-1.75 (m, 3H), 1.39-1.28 (2s, 9H). LCMS (ESI): m / z 236.0 [M ⁺ + 1-Boc]; HPLC: 76.79%; chiral HPLC: 96.26%

Synthesis of (S) -1- (tert-butoxycarbonyl) -2- (hydroxymethyl) pyrrolidine-2-carboxylic acid (6):
To a stirred solution of compound 5 (1.1 kg, 32.835 mol) in methanol (10 L) was added 50% wet 10% Pd-C (500 g) at room temperature and stirred for 8 h under H ₂ atmosphere (3 kg) . After starting material was consumed (by TLC), the reaction mixture was filtered through a pad of celite and the pad was washed with methanol (50 mL). The resulting filtrate was concentrated under reduced pressure to give a crude product, which was triturated with Et ₂ O to give compound 6 (650 g, 81%) as an off-white solid. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 3.92 to 3.80 (dd, 1 H), 3.64 to 3.61 (m, 1 H), 3.49 to 3.34 (m, m) 1H), 3.32 to 3.26 (m, 1H), 2.29 to 2.16 (m, 1H), 1.95 to 1.71 (m, 3H), 1.38 to 1.33 ( s, 9H); LCMS (ESI): m / z 243.8 [M ⁺ -1].

tert-Butyl (S) -2-(((2S, 3R) -1,3-bis (benzyloxy) -1-oxobutan-2-yl) carbamoyl) -2- (hydroxymethyl) pyrrolidine-1-carboxylate Synthesis of (7):
To a solution of compound 6 (450 g, 0.836 mol) in DCM (4.5 L) at 0-5 ° C. under nitrogen atmosphere was added N, N-diisopropylethylamine (846 mL, 4.591 mol). After stirring for 10 minutes, intermediate D1 (615 g, 1.836 mol) was added. After stirring for 10 minutes, HATU (701 g, 2.204 mol) was added. The reaction mixture was allowed to reach room temperature and stirred for 6 hours. After starting material was consumed (by TLC), the reaction mixture was diluted with DCM (1 L) and washed with water (2 × 2 L), 10% citric acid solution (2 L) and brine solution (2 L). The organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound which is purified by column chromatography eluting with 40% EtOAc / n-hexane to give compound 7 which Dissolve in Et ₂ O (1.5 L) and stir for 1 h. The resulting precipitate was filtered and dried under vacuum to give compound 7 (630 g, 65%) as a white solid. ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 7.97 to 7.89 (d, 0.5 H), 7.53 to 7.51 (d, 0.5 H), 7.32-7. 19 (m, 10 H), 5.64 (br s, 1 H), 5.16-5.04 (m, 2 H), 4.62-4.49 (m, 2 H), 4.31-4.28 (M, 1 H), 4.09-4.02 (m, 1.5 H), 3.87 (br s, 1 H), 3.57-3.56 (m, 0.5 H), 3.43- 3.37 (m, 1 H), 3.29-3. 25 (m, 1 H), 2.32-2.14 (m, 1 H), 1.98-1.90 (m, 1 H), 1. 75-1.69 (m, 2H), 1.33-1.26 (2s, 9H), 1.14-1.12 (d, 3H); LCMS (m / z): 527.6 [M ⁺ +1]; HPLC: 96. 0%.

tert-Butyl (S) -2-((2S, 3R) -1,3-bis (benzyloxy) -1-oxobutan-2-yl) -1-oxo-2,5-diazaspiro [3.4] octane Synthesis of -5-carboxylate (8):
To a solution of triphenylphosphine (295 g, 1.125 mol) in THF (846 mL) was added dropwise DIAD (227 g, 1.125 mol) at room temperature under a nitrogen atmosphere and stirred for 30 minutes. The reaction mixture was cooled to 15 ° C., and a solution of compound 7 (423 g, 0.804 mol) in THF (1.2 L) was added dropwise, and stirred at room temperature for 4 hours. The crude material obtained was washed with hexane and subsequently stirred with 50% Et ₂ O / hexane for 30 minutes. The precipitate formed was filtered off and the filtrate was concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography eluting with 40% EtOAc / hexane to give compound 8 (425 g, crude) as a brown syrup. ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 7.38-7.21 (m, 10 H), 5.17-5.10 (m, 2 H), 4.56-4.50 (m, 5) 2H), 4.31-4.28 (d, 1 H), 4.06-3.99 (m, 2 H), 3.89-3. 88 (d, 0.5 H), 3.48-3. 47 (d, 0.5 H), 3.33 to 3.34 (m, 1 H), 3.25 to 3.19 (m, 1 H), 2.10 to 2.02 (m, 2 H), 79-1.77 (m, 2H), 1.40-1.25 (s, 9H), 1.15-1.12 (d, 3H). LCMS (ESI): m / z 509.4 [M ⁺ +1]; HPLC: 92.31%; chiral HPLC: 86.47%.

(2S, 3R) -2-((S) -5- (tert-butoxycarbonyl) -1-oxo-2,5-diazaspiro [3.4] octan-2-yl) -3-hydroxybutanoic acid (9 Synthesis of):
A solution of compound 8 (283 g, 0.557 mol) in methanol (1.2 L) was degassed under a N ₂ atmosphere. Then, 50% wet 10% Pd-C (140 g) was added at room temperature and stirred for 24 hours under H ₂ atmosphere. After starting material was consumed (by TLC), the reaction mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The crude solid obtained was diluted with Et ₂ O (500 mL) and stirred vigorously at 0 ° C. for 1 h. The resulting solid was filtered off and dried to give compound 9 (140 g, 76%) as a white solid. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 12.86 (br s, 1 H), 5.24 (br s 1 H), 4.06-4.00 (m, 2 H), 3.88- 3.82 (m, 1 H), 3.51-3. 50 (m, 1 H), 3.43-3.24 (m, 1 H), 3.31-3. 23 (m, 1 H), 15-2.09 (m, 2H), 1.82-1.78 (m, 2H), 1.39-1.31 (2s, 9H), 1.10-1.08 (d, 3H); LCMS (m / z): 327.1 [M ⁺ -1]; HPLC: 95.44%; chiral HPLC: 100.00%.

tert-Butyl (S) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl) -1-oxo-2,5-diazaspiro [3.4] octane-5 -Synthesis of Carboxylate (B4):
A stirred solution of compound 9 (420 g, 1.28 mol) in CH ₂ Cl ₂ (4.2 L) at 0 ° C. under a nitrogen atmosphere HATU (584 g, 1.536 mol), NH ₄ Cl (137 g, 2.56 mol) And diisopropylethylamine (708 mL, 3.841 mol) were added. The reaction mixture was allowed to reach room temperature and stirred for 8 hours. After the starting material was consumed (by TLC), the reaction mixture was partitioned between water ( ₂ L) and CH ₂ Cl ₂ ( ₂ L) and stirred for 15 minutes. The organic layer was separated and washed with 2N HCl solution (2 × 1 L) and saturated NaHCO ₃ solution (2 L) and brine solution (2 L). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude product obtained was purified by silica gel column chromatography eluting with 5% MeOH / CH ₂ Cl ₂ to give compound B4 (320 g, mixture) as an off-white solid. The mixed compound B4 (87 g) was dissolved in DCM and reprecipitated with Et ₂ O with vigorous stirring for 1 h at room temperature. The product was filtered and dried under vacuum to give compound B4 (82 g) as a white solid. ¹ H-NMR: (400 MHz, D ₂ O): δ 4.53-4.10 (m, 2 H), 4.08-3.96 (m, 1 H), 3.80-3. 72 (m, 1 H) ), 3.60 to 3.39 (m, 2H), 2.53 to 2. 31 (m, 2H), 2.02 to 1.93 (m, 2H), 1.51 to 1.45 (s) , 9H), 1.31-1.29 (d, 3H); LCMS (ESI): m / z 326.1 [M ⁺ -1]; HPLC: 99.66%; chiral HPLC: 99. 76%; (C = 1, MeOH): -49.61.

Synthesis of (2S, 3R) -3-hydroxy-2-((S) -1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (10):
To a stirred solution of compound B4 (10 g, 0.031 mol) in CH ₂ Cl ₂ (40 mL) at 0 ° C. under nitrogen atmosphere was added TFA (23 mL, 0.305 mol). The reaction mixture was allowed to reach room temperature and stirred for 3 hours. After the starting material was consumed (by TLC), the volatiles were evaporated under reduced pressure. The resulting thick syrup material is washed with ether (2 × 50 mL) and dried under vacuum to give compound 10 (10 g, TFA salt, crude) as a thick syrup, which is taken to the next step Using. ¹ H-NMR: (400 MHz, D ₂ O): δ 4.38-4.10 (m, 2 H), 4.11-3. 94 (m, 2 H), 3.56-3.44 (m, 2 H) ), 2.52-2.38 (m, 2H), 2.24-2.16 (m, 2H), 1.39-1.27 (d, 3H); LCMS (ESI): m / z 228. 2 [M ⁺ +1].

Synthesis of (2S, 3R) -3-hydroxy-2-((S) -5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (compound B):
To a stirred solution of crude compound 10 (10 g, 0.029 mol TFA salt) in methanol (50 mL) isobutyraldehyde (6.5 mL, 0.073 mol) followed by AcOH (2.5 mL) at room temperature under a nitrogen atmosphere And stirred for 10 minutes. Then NaCNBH ₃ (5.4 g, 0.087 mol) was added in small portions and stirring was continued for 16 hours. Consumption of starting material (by TLC) was not complete. Again, isobutyraldehyde (3.2 mL, 0.036 mol) and AcOH (1 mL) were added and stirring was continued for 2 hours. After the starting material was consumed (by TLC), the reaction mixture was diluted with CH ₂ Cl ₂ (20 mL) and concentrated under reduced pressure. The crude material obtained is purified by silica gel column chromatography eluting with 4% MeOH / DCM to give (2S, 3R) -3-hydroxy-2-((S) -5-isobutyl-1-oxo-2, 5-diazaspiro [3.4] octan-2-yl) butanamide (6 g) was obtained as a thick syrup. ¹ H-NMR: (500 MHz, CD ₃ OD): δ 4.10-4.07 (m, 2 H), 3.63 (d, J = 5.5 Hz, 1 H), 3.49 (d, J = 6) .0 Hz, 1 H), 2.97-2.93 (m, 1 H), 2.74 (q, J = 8.0 Hz, 1 H), 2.41-2. 34 (m, 2 H), 2.26 -2.20 (m, 1 H), 2.17-2.11 (m, 1 H), 1.94-1.81 (m, 2 H), 1.73-1.69 (m, 1 H), 1 .21 (d, J = 6.0 Hz, 3 H), 0.91-0.89 (m, 6 H); LCMS (ESI): m / z 284.1 [M ⁺ +1]; HPLC: 99.53%; Chiral HPLC: 100.00%; SOR (c = 0.5, MeOH): -60.25.

(4S) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl) -5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octane- Synthesis of 5-ium (Z) -3-Carboxy Acrylate (Compound B Maleate):
(2S, 3R) -3-Hydroxy-2-((S) -5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (180 mg, 0.636 mmol) To the solution was added maleic acid (59 mg, 0.508 mmol) at room temperature and stirred for 16 hours. After the starting material was consumed (by TLC), the water was removed under reduced pressure. The crude material obtained is triturated with pentane and hexane, dried under vacuum and (4S) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl) -5-isobutyl-1-oxo-2,5-diazaspiro [3.4] octan-5-ium (Z) -3-carboxyacrylate (210 mg, 86%) was obtained as a pale yellow solid. ¹ H NMR (400 MHz, CD ₃ OD) δ 6.28 (s, 2 H), 4.21 to 4.11 (m, 2 H), 3.90 (d, J = 7.0 Hz, 1 H), 3.71 (D, J = 7.0 Hz, 1 H), 3.47-3.36 (m, 1 H), 3. 20-3.09 (m, 1 H), 2. 9-2. 82 (m, 1 H) , 2.81-2.71 (m, 1 H), 2.48-2.38 (m, 1 H), 2.36-2.26 (m, 1 H), 2.20-1. 3H), 1.24 (d, J = 6.0 Hz, 3 H), 1.00 (t, J = 7.0 Hz, 6 H); LCMS (ESI): m / z 284.2 [M ⁺ + 1-maleic acid HPLC: 97.98%; melting point: 114.8 ° C to 118.3 ° C.

Example 3 Synthesis of Compound C
Synthesis of (3R, 7aS) -3- (trichloromethyl) tetrahydro-1H, 3H-pyrrolo [1,2-c] oxazol-1-one (1):
To a suspension of L-proline (2.0 kg, 0.017 mol) in chloroform (50 L) at room temperature chloral hydrate (5.7 kg, 0.034 mol) was added. The reaction mixture was heated to 60 ° C. under a reverse Dean-Stark apparatus and the resulting water collected. After 16 hours, the volatiles were concentrated under reduced pressure. The crude solid was washed with cold ethanol, filtered and dried to give compound 1 (2.2 kg, 57%) as a white solid. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 5.82 (s, 1 H), 4.11 to 4.08 (m, 1 H), 3.33 to 3.27 (m, 2 H), 3 .19-3.14 (m, 1 H), 2.15-2.10 (m, 1 H), 1.96-1.91 (m, 1 H), 1.80-1.74 (m, 1 H) , 1.65-1.58 (m, 1 H).

Synthesis of (3R, 7aR) -7a-((benzyloxy) methyl) -3- (trichloromethyl) tetrahydro-1H, 3H-pyrrolo [1,2-c] oxazol-1-one (2):
N-BuLi (1.6 M in hexane) (958.5 mL, 1.533 mol) was added dropwise to a solution of diisopropylamine (221.2 mL, 1.533 mol) in THF (870 mL) at -78 C under a nitrogen atmosphere. After the addition was complete, the temperature of the reaction mixture was raised to -20.degree. C. and stirred for 1 hour. It was again cooled to −78 ° C., Compound 1 (250 g, 1.022 mol) in THF (1 L) was added and stirred for 30 minutes. Then, benzyl chloromethyl ether (208 mL, 1.329 mol) was added dropwise and stirring was continued for 1 hour. After the starting material had been consumed (by TLC), the reaction mixture was quenched with ice cold water (100 mL) and extracted with Et ₂ O (2 × 100 mL). The combined organic layers are washed with brine (100 mL), dried over anhydrous Na ₂ SO ₄ , concentrated under reduced pressure and purified by column chromatography eluting with 10% EtOAC / n-hexane to give a compound 2 (220 g, crude) was obtained as a brownish thick syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 7.42 to 7.26 (m, 5 H), 5.60 (s, 1 H), 4.59 to 4.53 (d, 2 H), 3 .67-3.61 (m, 2H), 3.37-3.33 (m, 1H), 3.31-3.10 (m, 1H), 2.12-1.99 (m, 2H) , 1.87-1.75 (m, 2H); LCMS (ESI): m / z 363.9 [M ⁺ +1].

Synthesis of methyl (R) -2-((benzyloxy) methyl) pyrrolidine-2-carboxylate hydrochloride (3):
To a solution of compound 2 (400 g, 1.096 mol) in methanol (1 L) at room temperature was added 2N HCl in MeOH (1.64 L, 3.29 mol) and stirred at 80 ° C. for 16 hours. After the starting material was consumed (by TLC), the volatiles were evaporated under reduced pressure. The crude product obtained was washed with hexane (3 × 750 mL) and dried under reduced pressure to give compound 3 (358 g, crude) as a reddish viscous syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 10.40 (br s, 1 H), 7.40 to 7.21 (m, 5 H), 4.64 to 4.50 (d, 2 H), 4.49-4.3 (d, 2 H), 3.76 (s, 3 H), 3.33-3.22 (m, 2 H), 2.22-2.15 (s, 1 H), 02-1.9 (m, 2H), 1.95 to 1.83 (m, 1H).

Synthesis of 1- (tert-Butyl) 2-methyl (R) -2-((benzyloxy) methyl) pyrrolidine-1,2-dicarboxylate (4):
To a stirred suspension of compound 3 (313 g, crude 1.096 mol) in DCM (2.19 L) at 0 ° C. under a nitrogen atmosphere Et ₃ N (458.4 mL, 3.288 mol) is added dropwise and stirred for 10 minutes did. Then Boc anhydride (358.4 g, 1.644 mol) was added dropwise at 0 ° C. The reaction mixture was allowed to reach room temperature and stirred for 16 hours. After starting material was consumed (by TLC), the reaction was diluted with water (2 × 1 L) and extracted with CH ₂ Cl ₂ (2 × 500 mL). The combined organic layers were washed with 10% citric acid (pH ̃7) and brine solution (1 L). The organic layer is dried over Na ₂ SO ₄ and concentrated under reduced pressure to give a crude compound which is purified by column chromatography eluting with 20% EtOAC / n-hexane to give compound 4 (215 g, 56%) Was obtained as a colorless viscous syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 7.37-7.26 (m, 5 H), 4.54-4.48 (m, 2 H), 4.03-3.87 (dd, 1 H), 3.69-3.67 (d, 1 H), 3.62 (s, 3 H), 3.53-3.47 (m, 1 H), 3.33-3.30 (m, 1 H) , 2.27-2.20 (m, 1 H), 2.03 to 1.89 (m, 2 H), 1.88 to 1.79 (m, 1 H), 1.46 to 1.24 (2 s, 2 s) 9 H); LCMS (ESI): m / z 250.1 [(M ⁺ +1) -Boc].

Synthesis of (R) -2-((benzyloxy) methyl) -1- (tert-butoxycarbonyl) pyrrolidine-2-carboxylic acid (5):
NaOH (73.9 g, 1.848 mol) was added to a solution of compound 4 (215 g, 0.616 mol) in MeOH: THF: H ₂ O (3 L, 5: 5: 3) and stirred at room temperature for 10 minutes. The reaction mixture was heated to 80 ° C. and stirred for 16 hours. After the starting material was consumed (by TLC), the reaction mixture was brought to room temperature and the volatiles were evaporated. The crude material was diluted with water (1 L) and extracted with Et ₂ O (2 × 500 mL). The separated aqueous layer was acidified with 2N HCl solution (pH ̃3) and extracted with DCM (2 × 750 mL). The combined organic layers were washed with brine solution, dried over Na ₂ SO ₄ and concentrated to give compound 5 (170 g, 82.4%) as an off-white solid. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 12.66 (br s, 1 H), 7.36 to 7.26 (m, 5 H), 4.54 to 4.47 (m, 2 H), 4.05 to 3.90 (dd, 1 H), 3.88 to 3.63 (d, 1 H), 3.63 to 3.44 (m, 1 H), 3.34 to 3.27 (m, 1 H) ), 2.27-2.01 (m, 1 H), 2.02-1.8 (m, 2 H), 1.79-1.76 (m, 1 H), 1.17-1.14 (2 s) , 9H); LCMS (ESI): m / z 235.1 [(M ⁺ +1) -Boc].

Synthesis of (R) -1- (tert-butoxycarbonyl) -2- (hydroxymethyl) pyrrolidine-2-carboxylic acid (6):
To a stirred solution of compound 5 (170 g, 0.507 mol) in CH ₃ OH (1 L) at room temperature was added 50% wet 10% Pd—C (68 g) and stirred under H ₂ atmosphere for 16 hours. After starting material was consumed (by TLC), the reaction mixture was filtered through a pad of celite and the pad was washed with CH ₃ OH (1 L). The resulting filtrate was concentrated under reduced pressure to give compound 6 (110 g, 88%) as a white solid. ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 12.66 (br s, 1 H), 3.96 to 3.83 (dd, 1 H), 3.63 to 3.60 (m, 1 H), 3.49-3.46 (m, 1 H), 3.34-3.25 (m, 2 H), 2.30-2.15 (m, 1 H), 1.95-1.72 (m, 3 H) LCS (ESI): m / z 244 [M ⁺ -1]; Chiral HPLC: 95. 88%.), 1.38-1.33 (2s, 9H);

tert-Butyl (R) -2-(((2S, 3R) -1,3-bis (benzyloxy) -1-oxobutan-2-yl) carbamoyl) -2- (hydroxymethyl) pyrrolidine-1-carboxylate Synthesis of (7):
To a stirred solution of compound 6 (110 g, 0.448 mol) in DCM (20 mL) reaction mixture cooled to 0 ° C. under N ₂ atmosphere was added diisopropylethylamine (206 mL, 1.12 mol), and intermediate D1 (150 g, 0.448 mol) and HATU (204 g, 0.537 mol) were added under nitrogen atmosphere. The reaction mixture was allowed to reach room temperature and stirred for 4 hours. After starting material was consumed (by TLC), the reaction mixture was diluted with DCM (1 L) and washed with water (2 × 500 mL), 10% citric acid solution (500 mL) and brine solution (500 mL). The organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give the crude compound which is purified by column chromatography eluting with 30% EtOAc / n-hexane to give compound 7 (143 g, 60% ) Was obtained as a colorless viscous syrup. ¹ H-NMR: (500 MHz, DMSO-d ₆ ): δ 8.20-8.12 (d, 1 H), 7.29-7.18 (m, 10 H), 5.83-5.59 (m, 1H), 5.08 (s, 2H), 4.50 to 4.44 (m, 2H), 4.30 to 4.26 (m, 1H), 4.08 to 4.00 (m, 2H) , 3.42 to 3.40 (m, 2 H), 3.39 to 3.29 (m, 1 H), 2.19 to 2.08 (m, 1 H), 1.96 to 1.87 (m, 1) 1H), 1.68-1.63 (m, 2H), 1.23-1.15 (2s, 9H), 1.4-1.13 (d, 3H); LCMS (m / z): 525 ^{.2 [M + -1]; HPLC} : 76.2%; chiral HPLC: 69.47%.

tert-Butyl (R) -2-((2S, 3R) -1,3-bis (benzyloxy) -1-oxobutan-2-yl) -1-oxo-2,5-diazaspiro [3.4] octane Synthesis of -5-carboxylate (8):
To a solution of triphenylphosphine (161.4 g, 0.612 mol) in THF (430 mL) was added dropwise DIAD (123.8 g, 0.612 mol) at room temperature under a nitrogen atmosphere and stirred for 15 minutes. A solution of compound 7 (215 g, 0.408 mol) in THF (860 mL) was added dropwise thereto, and the mixture was stirred at room temperature for 4 hours. After the starting material was consumed (by TLC), the volatiles were evaporated under reduced pressure. The crude material obtained was purified by silica gel column chromatography eluting with 20% EtOAc / hexane to give compound 8 (180 g, mixture with DIAD by-product) as a yellow syrup. ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 7.31 to 7.18 (m, 10 H), 5.18 to 5.10 (m, 2 H), 4.61 to 4.54 (m, 10) 2H), 4.27-4.18 (m, 2H), 3.78-3.77 (d, 1 H), 3.45-3.43 (d, 1 H), 3.35-3. 31 (m) d, 1H), 3.27-3.23 (m, 1H), 2.03-1.98 (m, 2H), 1.78-1.76 (m, 2H), 1.39-1. 31 (2s, 9H), 1.23-1.22 (d, 3H) (peak of DIAD by-product not captured); LCMS (ESI): m / z 509.4 [M ⁺ +1].

(2S, 3R) -2-((R) -5- (tert-butoxycarbonyl) -1-oxo-2,5-diazaspiro [3.4] octan-2-yl) -3-hydroxybutanoic acid (9 Synthesis of):
A solution of compound 8 (85 g, 0.167 mol) in methanol (850 mL) was degassed under a N ₂ atmosphere. Then 50% wet 10% Pd-C (40 g) was added at room temperature and stirred for 24 h under H ₂ atmosphere. After starting material was consumed (by TLC), the reaction mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The crude solid obtained was diluted with Et ₂ O (1 L) and stirred vigorously at room temperature for 1 h. The resulting solid was filtered off and dried to give compound 9 (75 g, 68%) as a white solid. ¹ H-NMR: (400 MHz, DMSO-d ₆ ): δ 12.86 (br s, 1 H), 5.24 (br s 1 H), 4.06-4.00 (m, 2 H), 3.88- 3.82 (m, 1 H), 3.51-3. 50 (m, 1 H), 3.43-3.24 (m, 1 H), 3.31-3. 23 (m, 1 H), 15-2.09 (m, 2H), 1.82-1.78 (m, 2H), 1.39-1.31 (2s, 9H), 1.10-1.08 (d, 3H); LCMS (m / z): 329.1 [M ⁺ +1]; HPLC: 93.97%.

tert-Butyl (R) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl) -1-oxo-2,5-diazaspiro [3.4] octane-5 -Synthesis of Carboxylate (A4):
A stirred solution of compound 9 (150 g, 0.457 mol) in CH ₂ Cl ₂ ( ₂ L) with the reaction mixture cooled to 0 ° C. to diisopropylethylamine (176.85 g, 1.37 mol), NH ₄ Cl (48. 8 g, 0.914 mol) and HATU (208.3 g 0.548 mol) were added under a nitrogen atmosphere. The reaction mixture was allowed to reach room temperature and stirred for 12 hours. After starting material was consumed (by TLC), the reaction mixture was diluted with water (2 × 750 mL) and extracted with CH ₂ Cl ₂ (2 × 1 L). The combined organic layers were washed with 2N HCl solution and saturated sodium chloride solution (750 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude compound was purified by column chromatography and pure compound was eluted with 3% MeOH / CH ₂ Cl ₂ . The resulting material was diluted with DCM (2 L) and washed with saturated citric acid (5 × 500 mL), saturated bicarbonate wash followed by brine wash and dried over Na ₂ SO ₄ . The material was triturated with ether (2 × 500 mL) to give compound A4 (80 g, 53%) as a white solid. ¹ H-NMR: (400 MHz, D ₂ O): δ 4.36 to 4.21 (m, 2 H), 4.03 to 3.98 (m, 1 H), 3.76 to 3.67 (m, 1 H) ), 3.56-. 3.37 (m, 2H), 2.32-2.23 (m, 2H), 1.97-1.93 (m, 2H), 1.49-1.47 (2s, 9H), 1. 33-1.32 (d, 3H); LCMS (ESI): m / z328.3 [M + +1]; HPLC: 99.30%; chiral HPLC: 98.97%; SOR (c = 1, CH 3 OH): −1.08.

Synthesis of (2S, 3R) -3-hydroxy-2-((R) -1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (10):
To a stirred solution of compound A4 (2 g, 6.16 mmol) in CH ₂ Cl ₂ (10 mL) at 0 ° C. under nitrogen atmosphere was added TFA (1.0 mL, 12.2 mmol). The reaction mixture was allowed to reach room temperature and stirred for 2 hours. After the starting material was consumed (by TLC), the volatiles were evaporated under reduced pressure. The resulting thick syrup material is washed with ether (2 × 50 mL) and dried under vacuum to give crude Compound 10 (2.5 g, TFA salt) as a thick syrup, which is the next step Used for ¹ H NMR (500 MHz, CD ₃ OD): δ 4.24-4.17 (m, 2 H), 3.99-3.89 (m, 2 H), 3.52-3. 39 (m, 2 H), 2.46-2.32 (m, 2H), 2.26-2.10 (m, 2H), 1.28 (d, J = 6.1 Hz, 3H); LCMS (ESI): m / z 228. 1 [M ⁺ +1].

(2S, 3R) -3-hydroxy-2-((R) -5-((S) -2-hydroxypropyl) -1-oxo-2,5-diazaspiro [3.4] octan-2-yl) Butanamide (Compound C):
A stirred solution of compound 10 (TFA salt, 2 g, 5.86 mmol) in methanol (10 mL) with NaOMe (630 mg, 11.7 mmol) followed by (S) -2-methyloxirane (510 mg, 8.79 mmol) with nitrogen Added at room temperature under atmosphere. The reaction mixture was stirred at room temperature for 24 hours. After the starting material was consumed (by TLC), the reaction mixture was diluted with CH ₂ Cl ₂ (10 mL) and concentrated under reduced pressure. The crude material obtained is purified by silica gel column chromatography eluting with 5% MeOH / DCM to give (2S, 3R) -3-hydroxy-2-((R) -5-((S) -2-hydroxy) Propyl) -1-oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (185 mg, 12%) was obtained as a thick syrup. ¹ H NMR (400 MHz, CD ₃ OD): δ 4.18-4.07 (m, 2 H), 3.90-3.81 (m, 1 H), 3.63 (d, J = 6.1 Hz, 1 H ), 3.58 (d, J = 6.1 Hz, 1 H), 3.11 (dt, J = 4.2, 8.2 Hz, 1 H), 2.83-2.67 (m, 2 H), 2 .62-2.54 (m, 1H), 2.26-2.12 (m, 2H), 1.98-1.83 (m, 2H), 1.24 (d, J = 6.1 Hz, 3H), 1.14 (d, J = 6.3 Hz, 3H); LCMS (ESI): m / z 285.9 [M ⁺ +1]; HPLC: 97.20%.

Example 4
This example shows a positive emotion learning (PEL) test. The experiment is described in Burgdorf et al. “The effect of selective breeding for differential rates of 50-kHz ultrasonic vocalisations on emotional behavior in rats,” Devel. Psychobiol. , 51: 34-46 (2009). The rat's 50 kHz ultrasound (Hedonic USV) is a valid model for the study of positive affective state and is best triggered by rough-and-tumble play. The 50 kHz ultrasound noise has a positive correlation with social behavior on reward and appetite in rats and has previously been shown to reflect a positive affect state.

  The PEL assay measures the acquisition of positive (hedonic) 50 kHz sonication (USV) to social stimuli, heterospecific conflicting play stimuli. Heterogeneous task-play play stimulation was given by the experimenter's right hand. The animals underwent three minutes of heterospecific play, consisting of alternating blocks of 15 sec blocks of heterospecific play and 15 sec blocks without stimulation. Burgdorf et al. , "Positive emotional learning is related in the medial frontal cortex by GluN2B-containing NMDA receptors," as previously described by Neuroscience, 192: 515-523 (2011), using Avasoft SASlab Pro (Germany). Sound wave high frequency ultrasound (USV) was recorded and analyzed. The PEL was determined by quantifying the frequency modulated 50 kHz USV generated during each non-stimulation period. The animals were not acclimatized prior to testing because they were irritating. Positive emotional learning was measured during conditional stimulus (CS) testing prior to tickling unconditional stimulus (UCS) testing. The animals underwent 15 second rounds of testing, each consisting of 6 CS and 6 UCS tests (total 3 minutes). At the end of the 3 minute session, the running speed of the animals for the tickling they received spontaneously was also measured.

  FIG. 1 shows the results of rat PEL test. The results demonstrate that Compound A, when administered 1 hour before the PEL test, increases the positive affect learning measured by rat USV, thereby exhibiting an antidepressant effect.

Example 5
The microsomal and plasma stability of Compound A was investigated. The following table shows the percent of Compound A remaining after 60 minutes.

Unbound drug concentrations in the brain were also investigated and plasma protein binding assays were performed. The following table (% bound) shows the results for compound A.

The bioavailability of Compound A was investigated after PO administration. CSF, brain and plasma samples were analyzed after administration of Compound A (10 mg / kg). The following table shows the bioavailability and the CSF / plasma and brain / plasma ratios.

Example 6
The assay is described by Moskal et al. “GLYX-13: a monoclonal antibody-derived peptide that acts as an N-methyl-D-aspartate receptor modulator,” as described by Neuropharmacology, 49, 1077-87, 2005. Enhanced binding of [ ³ H] MK-801 (5 nM; 22.5 Ci / mmol) to well washed rat cortical membranes (200 μg) in the presence of increasing concentrations of Compound A under non-equilibrium conditions Measured (15 minutes at 25 ° C.).
FIG. 2 shows the enhancement of [ ³ H] MK-801 binding of wild type NMDAR2 subtype to A. As shown in FIG. 2, compound A is highly preferred for NR2A and does not bind to NR2D.

Example 7
Sprague Dawley rats were intravenously administered 2 mg / kg of Compound A. Group 2 Sprague Dawley rats were dosed orally with 10 mg / kg of Compound A. Plasma samples were collected for 24 hours and analyzed for Compound A.
Figure 3 presents mean plasma concentration-time profiles of Compound A after single intravenous (2 mg / kg) and oral (10 mg / kg) doses in male Sprague Dawley rats over 24 hours.
In another experiment, Sprague Dawley rats were dosed orally with 10 mg / kg of Compound A. Plasma, brain and CSF samples were analyzed at various time points for 24 hours.
FIG. 4 shows mean plasma, brain and CSF concentration-time profiles of Compound A after a single oral (10 mg / kg) dose in male Sprague Dawley rats over 24 hours.

Example 8
This example relates to PEL experiments, as described above in Example 3. It is a rat ultrasound (USV) test.
Positive emotional learning was measured during conditional stimulus (CS) testing prior to tickling unconditional stimulus (UCS) testing. The animals underwent 15 second rounds of testing, each consisting of 6 CS and 6 UCS tests (total 3 minutes).
FIG. 5 shows the results of rat USV test. This result demonstrates that Compound B increases positive emotional learning in the rat USV test, thereby exhibiting an antidepressant effect.

Example 9
The microsomal and plasma stability of Compound B was investigated. The following table shows the percent of Compound B remaining after 60 minutes.

Unbound drug concentrations in the brain were also investigated and plasma protein binding assays were performed. The following table (% bound) shows the results for compound B.

The bioavailability of Compound B was investigated. CSF, brain and plasma samples were analyzed after administration of compound B. The following table shows the bioavailability and the CSF / plasma and brain / plasma ratios.

Compound A and Compound B can both reach CSF and brain at a higher rate, although they have lower oral bioavailability.

Example 10
The assay is described by Moskal et al. Conducted as described in Example 5 above according to (2005). Enhanced binding of [ ³ H] MK-801 (5 nM; 22.5 Ci / mmol) to well washed rat cortical membranes (200 μg) in the presence of increasing concentrations of Compound B under non-equilibrium conditions Measured (15 minutes at 25 ° C.). FIG. 6 shows enhanced [ ³ H] MK-801 binding to wild type NMDAR2 subtype of Compound B. Compound B is highly preferred for NR2B and NR2D, with lower potency at NR2A and NR2C.

Example 11
Sprague Dawley rats were dosed intravenously with 2 mg / kg of Compound B. Group 2 Sprague Dawley rats were dosed orally with 10 mg / kg of Compound B. Plasma samples were collected for 24 hours and analyzed for Compound B.
FIG. 7 shows mean plasma concentration-time profiles of Compound B after single intravenous (2 mg / kg) and oral (10 mg / kg) administration in male Sprague Dawley rats over 24 hours.
In another experiment, 10 mg / kg of Compound B was orally administered to Sprague Dawley rats. Plasma, brain and CSF samples were analyzed at various time points for 24 hours.
FIG. 8 shows mean plasma, brain and CSF concentration-time profiles of Compound B after a single oral (10 mg / kg) dose in male Sprague Dawley rats over 24 hours.

Example 12
An in vitro MN (micronucleus) assay was performed for compound A. The assay was also performed for Compound B. Cytotoxicity is shown as% of control growth. Cytotoxicity values less than 60% are labeled and the compounds are considered toxic at each concentration. The results of in vitro MN assay of Compound A are shown in FIG. The results of in vitro MN assay of Compound B are shown in FIG.
The Ames assay was performed for each of Compound A and Compound B. In the results of the Ames assay, a hyphen (-) indicates a negative result. If p <0.05, weak positives are indicated as "+". If p <0.01, strong positives are indicated as "++". If p <0.001, very strong positives are indicated as "+++". The results of Compound A Ames assay are shown in FIG. The results of the Ames assay for Compound B are shown in FIG.
The hERG assay was performed with Compound A to identify potential hERG channel interactions. In addition, hERG assay was performed using Compound B. Results showing greater than 50% inhibition or stimulation for hERG are considered to represent a significant effect on the test compound. The results of the hERG assay of Compound A are shown in FIG. 9, and the results of the hERG assay of Compound B are shown in FIG.

Example 13
Goldstein et al. “Chronic traumatic encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model,” Science Translational Medicine, Vol. 4, Issue 134, pp. A single blast-induced traumatic brain injury / cognitive disorder was induced for use in rats according to the protocol of 134ra 60, 2012. Two to three month old male Sprague Dawley rats were used. Rats were housed in Lucite cages with aspen wood chip backing and maintained on a 12:12 light / dark cycle (lights on at 5 am), with free access to Purina research chow (USA) and tap water throughout the study. Rats are first anesthetized using 3.5-4% isoflurane and then ears are blocked with a 1.5 x 1.5 mm foam plug (Pura-Fit®, Moldex-Metric Inc., Culver City, California) The rats were protected and placed in a head access rodent chest restraint (Stoelting, USA) to protect the body while allowing the head to move freely. Aluminum shock tubes (183 × 61 cm; L-3 Applied Technologies, USA) were placed 10 cm from the rat head. The rat was subjected to one blast of about 42 PSI of helium generated by piercing a 0.014 inch polyester film. A mock control was placed outside the blast radius. Animals are dosed with animals with Compound A (10 mg / kg PO), Compound B (10 mg / kg PO), or 0.5% Na-CMC vehicle (1 ml / kg PO) in 0.9% sterile saline 1 hour after blasting did.

PEL (Positive Emotion Learning) was conducted 48 hours after the blast. Heterospecific play has previously been described by Burgdorf et al. , “The long-lasting antidepressant effects of rappastinel (GLYX-13) are associated with a metaplasticity process in the medial frontal cortex and hippocampus,” as described in Neuroscience 308: 202-211, 2015. Heterogeneous task-play play stimulation was given by the experimenter's right hand. The animals underwent three minutes of heterospecific play, consisting of alternating blocks of 15 sec blocks of heterospecific play and 15 sec blocks without stimulation. Burgdorf et al. , "Positive emotional learning is related in the medial frontal cortex by GluN2B-containing NMDA receptors," as previously described by Neuroscience, 192: 515-523, 2011, using Avasoft SASlab Pro (Germany) to ultrasound High frequency ultrasound phonation (USV) was recorded and analyzed. The PEL was determined by quantifying the frequency modulated 50 kHz USV generated during each non-stimulation period. The animals were not acclimatized prior to testing because they were irritating.
The results of PEL studies for compounds A and B are reported in FIGS. 11 and 12.

Example 14
This example relates to the evaluation of the anti-anxiety effect of treatment in stress-induced anxiety models, novelty-induced anorexia. This assay is also sensitive to the fast acting antidepressant ketamine and GLYX-13 (Li et al., "MTOR-dependent synapse formations under the rapid antidepressant effects of NMDA antagonists," Science 329: 959-964, 2010; And Burgdorf et al., 2013). Bodnoff et al. See also, "A comparison of the effects of diazepam versus several typical and atypical anti-depressant drugs in an animal model of anxiety," Psychopharmacology, 97: 277-279, 1989.

  The test was performed as described in (Burgdorf et al., 2013). One version of the novel anorexia nervosa (NIH) test that has been previously shown to detect the acute anti-anxiety-like effect of ketamine (Li et al., 2010) was used. The animals were not fed overnight prior to testing, and the study diet was placed in the open chamber (40 x 40 x 20 cm) central chamber for 10 minutes under dark red illumination. Feces and urine were removed from the device between tests of each animal. After the NIH test, the waiting time for feeding in the animal's home cage was determined as a control to confirm that the effect on feeding was specific to the novel environment. The animals were videotaped and the waiting time for the first food intake (seconds) was manually scored off-line.

Single points per dose group that were 2 or more standard deviations from the mean were considered outliers and were excluded from data analysis. The ED ₅₀ was defined as the dose (or log linear interpolation of dose) showing half maximal effect (mean of vehicle and maximal effective dose) from the log (dose) linear (NIH value) plot. The maximum effective dose was defined as the lowest volume that was statistically different from vehicle but statistically equivalent to the higher dose (according to Fisher's PLSD; α set to 0.05).

  The results are shown in FIG. 13 and FIG. As shown in FIG. 13, Compound A produces an anxiolytic effect in the rat's novelty-induced anorexia (NIH) test. 2 treated with Compound A (10-1000 μg / kg PO; blue circles) or sterile saline + 0.5% Na-CMC vehicle (1 ml / kg PO; closed circles) 1 hour before a single 10-minute test session 2 Latency to feeding mean ± SEM in a novelty-induced anorexia (NIH) test in male SD rats of ̃3 months of age. The animals were not fed overnight prior to testing, and the study diet was placed in the central chamber of the open field during testing.

  As shown in FIG. 14, Compound B produces an anxiolytic effect in the rat's novel anorexia nervosa (NIH) test. Treated with Compound B (0.0001 to 1 μg / kg PO; blue circles) or sterile saline + 0.5% Na-CMC vehicle (1 ml / kg PO; closed circles) one hour before a single 10-minute test session Latency to eating in a novelty-induced anorexia (NIH) test in 2-3 month old male SD rats mean ± SEM. The animals were not fed overnight prior to testing, and the study diet was placed in the central chamber of the open field during testing.

Example 15
The analgesic efficacy of compounds measured by paw withdrawal threshold is assessed using the Bennett model of organic analgesia. Bennett GJ, Xie YK, "A peripheral mononeuropathy in rats that produces disorders of pain sensation like those seen in man," Pain 33: 87-107, 1988. If the animal recovers from surgery but still exhibits a low paw withdrawal threshold after application of von frey filaments, partial nerve injury surgery is performed on animals that are testing for analgesia response. Vehicle The animals undergo surgery and then receive the vehicle rather than the test compound. Animals were tested at 1, 24, and 1 week after test compound or vehicle administration.

  Two to three month old male Sprague Dawley rats were used. Harlan was a supplier of all studies. Rats were housed in Lucite cages with aspen wood chip backing and maintained on a 12:12 light / dark cycle (lights on at 5 am), with free access to Purina research chow (USA) and tap water throughout the study.

  Rats were anesthetized using inhaled isoflurane (2.5%). Partial nerve injury surgery was performed as previously described (Bennett and Xie, 1988). An incision (approximately 1.5 cm in length) was made parallel to and behind the thigh, on the back of the right hind leg skin with a scalpel blade. The biceps femoris muscle was separated from the femur muscle using a small apical hemostat. Common sciatic nerves were isolated and exposed using curved blunt head forceps. The entire sciatic nerve was ligated for an organic analgesia test. The nerve was loosely knotted using hemostatic forceps / forceps and chromic gut (5-0) and three ligations were placed on the nerve at 1 mm intervals. The ligature was tightened to the extent that the suture did not slip above or below the nerve. This protocol resulted in partial loss of function of the nerve. The common calcaneal and tibial branches of the sciatic nerve were ligated and cut for thermal analgesia testing, and the groin was preserved. See Decosterd I, Woolf CJ, "Spared nerve injury: an animal model of persistent peripheral neuropathic pain," Pain 87: 149-158, 2000. The test was performed 1-2 weeks after surgery.

  During the test, rats were acclimated for 15-20 minutes to the surface of a suspended wire mesh grid (1 cm × 1 cm, using a 0.3 cm diameter wire). Each Von Frey filament was pressed vertically from the smallest to the plantar surface of the injured (ipsilateral) hind foot until it bent slightly and then held for 6 seconds. The following larger filaments were used as well if no apparent hindfoot pull-in or tipping behavior was observed immediately after filament recovery. If there was a response, lower filaments were used. This was repeated until 6 responses were collected.

  Testing was performed 1 hour after Compound A and Compound B (0.1-10 mg / kg PO), gabapentin (150 mg / kg PO) or 0.5% Na-CMC in 0.9% sterile saline. The animals were retested 24 hours and 1 week after dosing. The results of Compound A are shown in FIG. 15, and the results of Compound B are shown in FIG. For both compounds, a single dose of 10 mg / kg PO produced analgesia in the Bennett model at 1 hour, 24 hours and 1 week post dose, but gabapentin only produces an analgesic effect for 1 hour after dose The

Example 16
A non-clinical in vivo pharmacological test (Porsolt assay) was performed to measure antidepressant-like effects. A negative control (0.5% sodium carboxymethylcellulose in 0.9% sterile saline) and a positive control (compound D) were used to compare to Compound A and Compound B. This study allowed the construction of dose response curves for the effect of each compound in the Porsolt forced swim test, which is assessed by the response of the rat during the 5 minute swim test (reduction in flotation time).

  Two to three month old male Sprague Dawley rats were used (Harlan, Indianapolis, IN). Rats were housed in Lucite cages with aspen wood chip backing and maintained on a 12:12 light / dark cycle (lights on at 5 am), with free access to Purina research chow (USA) and tap water throughout the study.

  The Porsolt forced swimming test adapted for use in rats is described by Burgdorf et al. (The long-lasting antidepressant effects of rapastinel (GLYX-13) are associated with a metaplasticity process in the median anterior cortex and hippocampus. Neuroscience 308: 202-211, 2015). The animals were placed in clear glass tubes 46 cm high x 20 cm diameter filled with tap water (23 ± 1 ° C.) for up to 30 cm for 15 minutes on the first day (acclimation), and 5 minutes on the subsequent day of testing. Animals were dosed 1 hour from administration of compound (compound A and compound B), positive control (compound D, 10 μg / kg) or vehicle (0.5% sodium carboxymethylcellulose (Na-CMC) in 0.9% sterile saline) It was tested later. I changed the water after every other animal. A float time, defined as the minimum amount of effort required to videotap the animal and keep the animal's head over the water, is blinded with high inter-rater reliability (Pearson's r> 0.9) Were scored off-line by the experimenter.

The EC ₅₀ was defined as the dose (or log linear interpolation of doses) showing half maximal effect (mean of vehicle and maximal effective dose) from a log (dose) linear plot. The maximum effective dose was defined as the lowest volume that was statistically different from vehicle but statistically equivalent (by Fisher's PLSD) to a dose higher than that dose.

  The results for compound A are shown in FIG. Compound A (10-1000 μg / kg PO; data points connected by blue circles or lines), positive control compound D (10 μg / kg PO; green circles or 10 μg / kg PO data points) or in adult male Sprague Dawley rats Na-CMC (1 ml / kg PO; filled circles or 1 ml / kg PO data points) in 0.9% saline was administered 1 hour prior to testing. The mean ± SEM floating time in rat Porsolt test is shown. The animals received a 15 minute acclimation session one day before the 5 minute test. It is N = 6-18 per group. As shown, Compound A produces an antidepressant-like effect as measured by the decrease in suspension time in the Porsolt test, as compared to the vehicle group one hour after administration.

  The results for compound B are shown in FIG. Compound B (0.1-10 μg / kg PO; data points connected by blue circles or lines), positive control compound D (10 μg / kg PO; green circles or 10 μg / kg PO data points in adult male Sprague Dawley rats) ) Or 0.9% saline in Na-CMC (1 ml / kg PO; filled circles or 1 ml / kg PO data points) were administered 1 hour prior to testing. The mean ± SEM floating time in rat Porsolt test is shown. The animals received a 15 minute acclimation session one day before the 5 minute test. It is N = 6-18 per group. As shown, Compound B produces an antidepressant-like effect as measured by the decrease in suspension time in the Porsolt test, as compared to the vehicle group one hour after administration.

Example 17
The table below shows compound A, compound B, tert-butyl (R) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl) -1-oxo-2, 5-diazaspiro [3.4] octane-5-carboxylate (compound W), tert-butyl (S) -2-((2S, 3R) -1-amino-3-hydroxy-1-oxobutan-2-yl ) -1-Oxo-2,5-diazaspiro [3.4] octane-5-carboxylate (Compound X), (2S, 3R) -3-hydroxy-2-((R) -5-isobutyryl-1-) Oxo-2,5-diazaspiro [3.4] octan-2-yl) butanamide (compound Y) or (2S, 3R) -3-hydroxy-2-((S) -5-isobutyryl-1-oxo- 2,5-Diazaspiro 3.4] octane-2-yl) butanamide (showing the relative in vivo binding data wildtype NMDAR2 subtype in the presence of the compound Z).

Equivalents One of ordinary skill in the art can recognize or ascertain, using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Incorporation by Reference The entire contents of the patents, published patent applications, websites and other references cited herein are expressly incorporated herein by reference in their entirety.

Claims (10)

Formula I:
(I)
Wherein R ^b is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano and C ₁ -C ₆ alkyl,
R ¹ is selected from the group consisting of hydrogen, halogen and C ₁ -C ₆ alkyl, and R ² is selected from the group consisting of hydrogen, halogen and C ₁ -C ₆ alkyl)
Or a stereoisomer, N-oxide and / or a pharmaceutically acceptable salt thereof. The compound according to claim 1, wherein R ^b is hydrogen. The compound according to claim 1 or 2, wherein R ¹ is hydrogen. The compound according to any one of claims 1 to 3, wherein R ² is hydrogen. formula:
Or a stereoisomer, N-oxide and / or a pharmaceutically acceptable salt thereof. formula:
The compound according to claim 5, or an N-oxide and / or a pharmaceutically acceptable salt thereof, which has formula:
The compound according to claim 5, having N or an N-oxide or a pharmaceutically acceptable salt thereof.   A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.   The pharmaceutical composition according to claim 8, which is suitable for oral administration.   9. The pharmaceutical composition according to claim 8, which is suitable for intravenous, intranasal, subcutaneous and / or sublingual administration.